EGFR and c-Met fibronectin type III domain binding molecules

ABSTRACT

Monospecific and bispecific EGFR and/or c-Met FN3 domain containing molecules, isolated nucleotides encoding the molecules, vectors, host cells, and methods of making thereof are useful in the generation of therapeutic molecules and treatment and diagnosis of diseases and disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/086,250, filed 21 Nov. 2013, which claims the benefit of U.S.Provisional Application No. 61/728,906, filed 21 Nov. 2012, U.S.Provisional Application No. 61/728,914, filed 21 Nov. 2012, U.S.Provisional Application No. 61/728,912, filed 21 Nov. 2012, U.S.Provisional Application No. 61/782,550, filed 14 Mar. 2013 and U.S.Provisional Application No. 61/809,541, filed 8 Apr. 2013, the entirecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to monospecific or bispecific EGFR and/orc-Met binding molecules and methods of making and using the molecules.

BACKGROUND OF THE INVENTION

Epidermal growth factor receptor (EGFR, ErbB1 or HER1) is atransmembrane glycoprotein of 170 kDa that is encoded by the c-erbB1proto-oncogene. EGFR is a member of the human epidermal growth factorreceptor (HER) family of receptor tyrosine kinases (RTK) which includesHER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). EGFR signaling is initiatedby ligand binding followed by induction of conformational change,homodimerization or heterodimerization of the receptor with other ErbBfamily members, and trans-autophosphorylation of the receptor (Fergusonet al., Annu Rev Biophys, 37: 353-73, 2008), which initiates a signaltransduction cascades that ultimately affects a wide variety of cellularfunctions, including cell proliferation and survival. Increases inexpression or kinase activity of EGFR have been linked with a range ofhuman cancers, making EGFR an attractive target for therapeuticintervention (Mendelsohn et al., Oncogene 19: 6550-6565, 2000; GrÜnwaldet al., J Natl Cancer Inst 95: 851-67, 2003; Mendelsohn et al., SeminOncol 33: 369-85, 2006). Increases in both the EGFR gene copy number andprotein expression have been associated with favorable responses to theEGFR tyrosine kinase inhibitor, IRESSA™ (gefitinib), in non-small celllung cancer (Hirsch et al., Ann Oncol 18:752-60, 2007).

EGFR therapies include both small molecules and anti-EGFR antibodies,approved for treatment of colorectal cancer, pancreatic cancer, head andneck cancer, and non-small cell lung cancer (NSCLC) (Baselga andArteaga, J Clin Oncol 23:2445-2459 (20005; Gill et al., J Biol Chem,259:7755-7760, 1984; Goldstein et al., Clin Cancer Res, 1:131 1-1318;1995; Prewett et al., Clin Cancer Res, 4:2957-2966, 1998).

Efficacy of anti-EGFR therapies may depend on tumor type and EFGRmutation/amplification status in the tumor. Side effects of currenttherapeutics may include skin toxicity (De Roock et al., Lancet Oncol11:753-762, 2010; Linardou et al., Nat Rev Clin Oncol, 6: 352-366, 2009;Li and Perez-Soler, Targ Oncol 4: 107-119, 2009). EGFR tyrosine kinaseinhibitors (TKI) are commonly used as 2^(nd) line therapies for nonsmall cell lung cancer (NSCLC), but often stop working within twelvemonths due to resistance pathways (Riely et al., Clin Cancer Res 12:839-44, 2006).

c-Met encodes a tyrosine kinase receptor. It was first identified as aproto-oncogene in 1984 after it was found that treatment with acarcinogen resulted in a constitutively active fusion protein TPR-MET(Cooper et al., Nature 311:29-33, 1984). Activation of c-Met by itsligand hepatocyte growth factor (HGF) stimulates a plethora of cellprocesses including growth, motility, invasion, metastasis,epithelial-mesenchymal transition, angiogenesis/wound healing, andtissue regeneration (Christensen et al., Cancer Lett 225:1-26, 2005;Peters and Adjei, Nat Rev Clin Oncol 9:314-26, 2012). c-Met issynthesized as a single chain protein that is proteolytically cleavedinto a 50 kDa alpha- and 140 kDa beta-subunit linked by a disulphidebond (Ma et al., Cancer and Metastasis Reviews, 22: 309-325, 2003).c-Met is structurally similar to other membrane receptors such as Ronand. The exact stoichiometry of HGF:c-Met binding is unclear, but it isgenerally believed that two HGF molecules bind to two c-Met moleculesleading to receptor dimerization and autophosphorylation at tyrosines1230, 1234, and 1235 (Stamos et al., The EMBO Journal 23: 2325-2335,2004). Ligand-independent c-Met autophosphorylation can also occur dueto gene amplification, mutation or receptor over-expression.

c-Met is frequently amplified, mutated or over-expressed in many typesof cancer including gastric, lung, colon, breast, bladder, head andneck, ovarian, prostate, thyroid, pancreatic, and CNS cancers. Missensemutations typically localized to the kinase domain are commonly found inhereditary papillary renal carcinomas (PRCC) and in 13% of sporadicPRCCs (Schmidt et al., Oncogene 18: 2343-2350, 1999). c-Met mutationslocalized to the semaphorin or juxtamembrane domains of c-Met arefrequently found in gastric, head and neck, liver, ovarian, NSCLC andthyroid cancers (Ma et al., Cancer and Metastasis Reviews, 22: 309-325,2003; Sakakura et al., Chromosomes and Cancer, 1999. 24:299-305). c-Metamplification has been detected in brain, colorectal, gastric, and lungcancers, often correlating with disease progression (Ma et al., Cancerand Metastasis Reviews, 22: 309-325, 2003). Up to 4% and 20% ofnon-small cell lung cancer (NSCLC) and gastric cancers, respectively,exhibit c-Met amplification (Sakakura et al., Chromosomes and Cancer,1999. 24:299-305: Sierra and Tsao, Therapeutic Advances in MedicalOncology, 3:S21-35, 2011). Even in the absence of gene amplification,c-Met overexpression is frequently observed in lung cancer (Ichimura etal., Jpn J Cancer Res, 87:1063-9, 1996). Moreover, in clinical samples,nearly half of lung adenocarcinomas exhibited high levels of c-Met andHGF, both of which correlated with enhanced tumor growth rate,metastasis and poor prognosis (Sierra and Tsao, Therapeutic Advances inMedical Oncology, 3:S21-35, 2011; Siegfried et al., Ann Thorac Surg 66:1915-8, 1998).

Nearly 60% of all tumors that become resistant to EGFR tyrosine kinaseinhibitors increase c-Met expression, amplify c-Met, or increase itsonly known ligand, HGF (Turke et al., Cancer Cell, 17:77-88, 2010),suggesting the existence of a compensatory pathway for EGFR throughc-Met. c-Met amplification was first identified in cultured cells thatbecame resistant to gefinitib, an EGFR kinase inhibitor, and exhibitedenhanced survival through the Her3 pathway (Engelman et al., Science,316:1039-43, 2007). This was further validated in clinical samples wherenine of 43 patients with acquired resistance to either erlotinib orgefitinib exhibited c-Met amplification, compared to only two of 62untreated patients. Four of the nine treated patients also acquired theEGFR activating mutation, T790M, demonstrating simultaneous resistancepathways (Beat et al., Proc Natl Acad Sci USA, 104:20932-7, 2007).

The individual roles of both EGFR and c-Met in cancer is wellestablished, making these targets attractive for combination therapy.Both receptors signal through the same survival and anti-apoptoticpathways (ERK and AKT); thus, inhibiting the pair in combination maylimit the potential for compensatory pathway activation therebyimproving overall efficacy. Combination therapies targeting EGFR andc-Met are tested in clinical trials with Tarceva® (erlotinib) incombination with anti-c-Met monovalent antibody for NSCL (Spigel et al.,2011 ASCO Annual Meeting Proceedings 2011, Journal of Clinical Oncology:Chicago, Ill. p. 7505) and Tarceva (erlotinib) in combination withARQ-197, a small molecule inhibitor of c-Met (Adjei et al., Oncologist,16:788-99, 2011). Combination therapies or bispecific anti-EGFR/c-Metmolecules have been disclosed for example in: Int. Pat. Publ. No.WO2008/127710, U.S. Pat. Publ. No. US2009/0042906, Int. Pat. Publ. No.WO2009/111691, Int. Pat. Publ. No. WO2009/126834, Int. Pat. Publ. No.WO2010/039248, Int. Pat. Publ. No. WO2010/115551.

Current small molecule and large molecule therapeutic approaches toantagonize EGFR and/or c-Met signaling pathways for therapy may besub-optimal due to possible lack of specificity, potential off-targetactivity and dose-limiting toxicity that may be encountered with smallmolecule inhibitors. Typical bivalent antibodies may result inclustering of membrane bound receptors and unwanted activation of thedownstream signaling pathways. Monovalent antibodies (half arms) posesignificant complexity and cost to the manufacturing process.

Accordingly, the need exists for additional monospecific and bispecificEGFR and/or c-Met inhibitors for both therapeutic and diagnosticpurpose.

SUMMARY OF THE INVENTION

One aspect of the invention is an isolated bispecific FN3 domaincontaining molecule comprising a first fibronectin type III (FN3) domainand a second FN3 domain, wherein the first FN3 domain specifically bindsepidermal growth factor receptor (EGFR) and blocks binding of epidermalgrowth factor (EGF) to EGFR, and the second FN3 domain specificallybinds hepatocyte growth factor receptor (c-Met), and blocks binding ofhepatocyte growth factor (HGF) to c-Met.

Another aspect of the invention is an isolated bispecific FN3 domaincontaining molecule comprising a first fibronectin type III (FN3) domainand a second FN3 domain wherein the first FN3 domain comprises an aminoacid sequence at least 87% identical to the amino acid sequence of SEQID NO: 27, and the second FN3 domain comprises an amino acid sequence atleast 83% identical to the amino acid sequence of SEQ ID NO: 41.

In other embodiments, the invention provides for bispecific EGFR/c-Metbinding and monospecific EGFR or c-Met binding FN3 domain containingmolecules having certain sequences.

Another aspect of the invention is an isolated fibronectin type III(FN3) domain that specifically binds epidermal growth factor receptor(EGFR) and blocks binding of epidermal growth factor (EGF) to EGFR,wherein the FN3 domain is isolated from a library designed based on theTencon amino acid sequence of SEQ ID NO: 1.

Another aspect of the invention is an isolated fibronectin type III(FN3) domain that specifically binds hepatocyte growth factor receptor(c-Met) and blocks binding of hepatocyte growth factor (HGF) to c-Met.

Another aspect of the invention is an isolated polynucleotide encodingthe molecule of the invention. Another aspect of the invention is avector comprising the polynucleotide of the invention.

Another aspect of the invention is a host cell comprising the vector ofthe invention

Another aspect of the invention is a method of producing a bispecificmolecule, comprising culturing the isolated host cell of the inventionunder conditions such that the bispecific molecule is expressed, andpurifying the bispecific molecule.

Another aspect of the invention is a pharmaceutical compositioncomprising the molecule of the invention and a pharmaceuticallyacceptable carrier.

Another aspect of the invention is a method of treating a subject havingcancer, comprising administering a therapeutically effective amount ofthe bispecific EGFR/c-Met FN3 domain containing molecule or the EGFR orc-Met binding FN3 domain to a patient in need thereof for a timesufficient to treat cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Amino acid alignment of the EGFR-binding FN3 domains.The BC and FG loops are boxed at residues 22-28 and 75-86 of SEQ ID NO:18. Some variants include thermal stability improving L17A, N46K andE86I substitutions (residue numbering according to Tencon SEQ ID NO: 1).P54AR4-83v2 (SEQ ID NO: 27) paratope residues are underlined (D23, F27,Y28, V77, G85 in SEQ ID NO: 27).

FIG. 2. Sequence alignment of the Tencon27 scaffold (SEQ ID NO: 99) anda TCL14 library (SEQ ID NO: 100) having randomized C-CD-F-FG alternativesurface. The loop residues are boxed. Loops and strands are indicatedabove the sequences.

FIG. 3. Sequence alignment of the c-Met-binding FN3 domains. The C loopand the CD strand and the F loop and the FG strand are boxed and spanresidues 29-43 and 65-81. P114AR7P95-A3 (SEQ ID NO: 41) paratoperesidues are underlined (R34S, F38S, M72S and I79S).

FIG. 4. Inhibition of c-Met phosphorylation in H292 cells pre-treatedwith monospecific or bispecific FN3 domain containing molecules andstimulated with HGF is shown. Substantial increase in the potency of thebispecific EGFR/c-Met molecule (ECB1) was observed when compared to amonospecific c-Met-binding FN3 domain (P114AR5P74-A5, shown as A5 in theFigure) on its own or in combination with an EGFR-binding FN3 domain(P54AR4-83v2, shown as 83v2 in the Figure).

FIG. 5. Inhibition of EGFR and c-Met phosphorylation in cellspre-treated with monospecific or bispecific FN3 domain containingmolecules. In cell lines expressing high levels of EGFR, NCI-H292 (FIG.5A) and H596 FIG. 5(B), anti-EGFR monospecific and bispecific FN3 domaincontaining molecules are equally potent at decreasing EGFRphosphorylation. In cell lines expressing low levels of EGFR relative toc-Met, H441 (FIG. 5C), bispecific EGFR/c-Met molecules improve thepotency for inhibition of EGFR phosphorylation compared to themonospecific EGFR-binding FN3 domain alone. In cell lines with lowlevels of c-Met, relative to EGFR, H292 (FIG. 5D) and H596 (FIG. 5E),inhibition of c-Met phosphorylation is significantly potentiated withbispecific EGFR/c-Met molecule, compared to monospecific c-Met-bindingFN3 domain only. Molecules used in the study were: bispecific ECB5(shown as 17-A3 in the Figure), monospecific EGFR-binding FN3 domainP53A1R5-17 (shown as “17” in the Figure), bispecific EGFR/c-Met moleculeECB3 (shown as 83-H9 in the Figure), and monospecific c-Met binding FN3domain P114AR7P93-H9 (shown as H9 in the Figure).

FIG. 6. Pharmacodynamic signaling in tumors isolated from mice dosedwith bispecific EGFR/c-Met molecules for 6 h or 72 h. All moleculessignificantly reduced c-Met, EGFR and ERK phosphorylation at both 6 hand 72 h, the degree if inhibition was dependent on the affinity of theFN3 domains to EGFR and/or c-Met. Bispecific molecules were generated byjoining EGFR-binding FN3 domain with a high (“83” in the Figure isp54AR4-83v2) or medium (“17v2” in the Figure is P53A1R5-17v2) affinityto a c-Met-binding FN3 domain with high (“A3” in the Figure isP114AR7P94-A3) or medium (“A5” in the Figure is P114AR5P74-A5) affinity.

FIG. 7: Plasma (top) and tumor (bottom) accumulation of bispecificEGFR/cMet molecules of variable affinities linked to an albumin bindingdomain (ABD) are shown 6 h (left) and 72 h (right) after IP dosing. Sixhours after dosing, tumor accumulation is maximal in mice dosed with abispecific molecule harboring a medium affinity EGFR-binding FN3 domain(17v2) or high affinity EGFR binding domain (83v2). The bispecificmolecules incorporated high or medium affinity EGFR or c-Met binding FN3domains as follows: 83v2-A5-ABD (ECB18; high/medium for EGFR/cMet)83v2-A3-ABD (ECB38; high/high) 17v2-A5 (ECB28; medium/medium)17v2-A3-ABD (ECB39; medium/high). In the figure, 83v2 refers top54AR4-83v2; 17v2 refers to p53A1R5-17v2; A3 refers to p114AR7P94-A3 andA5 refers to p114AR5P74-A5.

FIG. 8. H292-HGF tumor xenografts were implanted into SCID beige mice.When tumors reached an average volume of approximately 80 mm³, mice weredosed three times per week with bispecific EGFR/c-Met molecules (25mg/kg) or PBS vehicle. All bispecific molecules reduced tumor growth,the tumor growth inhibition (TGI) being dependent on the affinities ofthe molecules for c-Met and EGFR. (high EGFR-high cMet refers top54AR4-83v2-p114AR7P94-A3 (ECB38); high EGFR-med cMet refers top54AR4-83v2-p114AR5P74-A5 (ECB18); med EGFR-high cMet refers top53A1R5-17v2-p114AR7P94-A3 (ECB39); med EGFR-med-cMet refers top53A1R5-17-p114AR5P74-A 5 (ECB28)).

FIG. 9. H292-HGF tumor xenografts were implanted into SCID beige miceand they were treated with different therapies. The anti-tumor activityof the therapies is shown. (bispecific EGFR/c-Met molecule refers top54AR4-83v2-p114AR7P94-A3-ABD (ECB38); the other therapies arecrizotinib, erlotinib, cetuximab, and the combination of crizotinib anderlotinib).

DETAILED DESCRIPTION OF THE INVENTION

The term “fibronectin type III (FN3) domain” (FN3 domain) as used hereinrefers to a domain occurring frequently in proteins includingfibronectins, tenascin, intracellular cytoskeletal proteins, cytokinereceptors and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad SciUSA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993;Watanabe et al., J Biol Chem 265:15659-15665, 1990). Exemplary FN3domains are the 15 different FN3 domains present in human tenascin C,the 15 different FN3 domains present in human fibronectin (FN), andnon-natural synthetic FN3 domains as described for example in U.S. Pat.Publ. No. 2010/0216708. Individual FN3 domains are referred to by domainnumber and protein name, e.g., the 3^(th) FN3 domain of tenascin (TN3),or the 10^(th) FN3 domain of fibronectin (FN10).

The term “substituting” or “substituted” or ‘mutating” or “mutated” asused herein refers to altering, deleting of inserting one or more aminoacids or nucleotides in a polypeptide or polynucleotide sequence togenerate a variant of that sequence.

The term “randomizing” or “randomized” or “diversified” or“diversifying” as used herein refers to making at least onesubstitution, insertion or deletion in a polynucleotide or polypeptidesequence.

“Variant” as used herein refers to a polypeptide or a polynucleotidethat differs from a reference polypeptide or a reference polynucleotideby one or more modifications for example, substitutions, insertions ordeletions.

The term “specifically binds” or “specific binding” as used hereinrefers to the ability of the FN3 domain of the invention to bind to apredetermined antigen with a dissociation constant (K_(D)) of about1×10⁻⁶ M or less, for example about 1×10⁻⁷ M or less, about 1×10⁻⁸ M orless, about 1×10⁻⁹ M or less, about 1×10⁻¹⁰ M or less, about 1×10⁻¹¹ Mor less, about 1×10⁻¹² M or less, or about 1×10⁻¹³ M or less. Typicallythe FN3 domain of the invention binds to a predetermined antigen (i.e.EGFR or c-Met) with a K_(D) that is at least ten fold less than itsK_(D) for a nonspecific antigen (for example BSA or casein) as measuredby surface plasmon resonance using for example a Proteon Instrument(BioRad). Thus, a bispecific EGFR/c-Met FN3 domain containing moleculeof the invention specifically binds to each EGFR and c-Met with abinding affinity (K_(D)) of at least 1×10⁻⁶ M or less for both EGFR andc-Met. The isolated FN3 domain of the invention that specifically bindsto a predetermined antigen may, however, have cross-reactivity to otherrelated antigens, for example to the same predetermined antigen fromother species (homologs).

The term “library” refers to a collection of variants. The library maybe composed of polypeptide or polynucleotide variants.

The term “stability” as used herein refers to the ability of a moleculeto maintain a folded state under physiological conditions such that itretains at least one of its normal functional activities, for example,binding to a predetermined antigen such as EGFR or c-Met.

“Epidermal growth factor receptor” or “EGFR” as used here refers to thehuman EGFR (also known as HER-1 or Erb-B1 (Ullrich et al., Nature309:418-425, 1984) having the sequence shown in SEQ ID NO: 73 and inGenBank accession number NP_005219, as well as naturally-occurringvariants thereof. Such variants include the well known EGFRvIII andother alternatively spliced variants (e.g., as identified by SwissProtAccession numbers P00533-1 (wild type; identical to SEA ID NO: 73 andNP_005219), P00533-2 (F404L/L4055), P00533-3 (628-705: CTGPGLEGCP . . .GEAPNQALLR→PGNESLKAML . . . SVIITASSCH and 706-1210 deleted), P00533-4(C628S and 629-1210 deleted), variants Q98, R266, K521, I674, G962, andP988 (Livingston et al., NIEHS-SNPs, environmental genome project, NIEHSES15478), T790M, L858R/T790M and del(E746, A750).

“EGFR ligand” as used herein encompasses all (e.g., physiological)ligands for EGFR, including EGF, TGF-α, heparin binding EGF (HB-EGF),amphiregulin (AR), and epiregulin (EPI).

“Epidermal growth factor” (EGF) as used herein refers to the well known53 amino acid human EGF having an amino acid sequence shown in SEQ IDNO: 74.

“Hepatocyte growth factor receptor” or “c-Met” as used herein refers tothe human c-Met having the amino acid sequence shown in SEQ ID NO: 101or in GenBank Accession No: NP_001120972 and natural variants thereof.

“Hepatocyte growth factor” (HGF) as used herein refers to the well knownhuman HGF having the amino acid sequence shown in SEQ ID NO: 102 whichis cleaved to form a dimer of an alpha and beta chain linked by adisulfide bond.

“Blocks binding” or “inhibits binding”, as used herein interchangeablyrefers to the ability of the FN3 domains of the invention of thebispecific EGFR/c-Met FN3 domain containing molecule to block or inhibitbinding of the EGFR ligand such as EGF to EGFR and/or HGF to c-Met, andencompass both partial and complete blocking/inhibition. Theblocking/inhibition of EGFR ligand such as EGF to EGFR and/or HGF toc-Met by the FN3 domain or the bispecific EGFR/c-Met FN3 domaincontaining molecule of the invention reduces partially or completely thenormal level of EGFR signaling and/or c-Met signaling when compared tothe EGFR ligand binding to EGFR and/or HGF binding to c-Met withoutblocking or inhibition. The FN3 domain or the bispecific EGFR/c-Met FN3domain containing molecule of the invention “blocks binding” of the EGFRligand such as EGF to EGFR and/or HGF to c-Met when the inhibition is atleast 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Inhibition ofbinding can be measured using well known methods, for example bymeasuring inhibition of binding of biotinylated EGF on EGFR expressingA431 cells exposed to the FN3 domain or the bispecific EGFR/c-Met FN3domain containing molecule of the invention using FACS, and usingmethods described herein, or measuring inhibition of binding ofbiotinylated HGF on c-Met extracellular domain using well known methodsand methods described herein.

The term “EGFR signaling” refers to signal transduction induced by EGFRligand binding to EGFR resulting in autophosphorylation of at least onetyrosine residue in the EGFR. An exemplary EGFR ligand is EGF.

“Neutralizes EGFR signaling” as used herein refers to the ability of theFN3 domain of the invention to inhibit EGFR signaling induced by EGFRligand such as EGF by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100%.

The term “c-Met signaling” refers to signal transduction induced by HGFbinding to c-Met resulting in autophosphorylation of at least onetyrosine residue in the c-Met. Typically at least one tyrosine residueat positions 1230, 1234, 1235 or 1349 is autophosphorylated upon HGFbinding.

“Neutralizes c-Met signaling” as used herein refers to the ability ofthe FN3 domain of the invention to inhibit c-Met signaling induced byHGF by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

“Overexpress”, “overexpressed” and “overexpressing” as used hereininterchangeably refer to a cancer or malignant cell that has measurablyhigher levels of EGFR and/or c-Met on the surface compared to a normalcell of the same tissue type. Such overexpression may be caused by geneamplification or by increased transcription or translation. EGFR and/orc-Met expression and overexpression can be measured using well knowassays using for example ELISA, immunofluorescence, flow cytometry orradioimmunoassay on live or lysed cells. Alternatively, or additionally,levels of EGFR and/or c-Met-encoding nucleic acid molecules may bemeasured in the cell for example using fluorescent in situhybridization, Southern blotting, or PCR techniques. EGFR and/or c-Metis overexpressed when the level of EGFR and/or c-Met on the surface ofthe cell is at least 1.5-fold higher when compared to the normal cell.

“Tencon” as used herein refers to the synthetic fibronectin type III(FN3) domain having the sequence shown in SEQ ID NO: 1 and described inU.S. Pat. Publ. No. US2010/0216708.

A “cancer cell” or a “tumor cell” as used herein refers to a cancerous,pre-cancerous or transformed cell, either in vivo, ex vivo, and intissue culture, that has spontaneous or induced phenotypic changes thatdo not necessarily involve the uptake of new genetic material. Althoughtransformation can arise from infection with a transforming virus andincorporation of new genomic nucleic acid, or uptake of exogenousnucleic acid, it can also arise spontaneously or following exposure to acarcinogen, thereby mutating an endogenous gene. Transformation/canceris exemplified by, e.g., morphological changes, immortalization ofcells, aberrant growth control, foci formation, proliferation,malignancy, tumor specific markers levels, invasiveness, tumor growth orsuppression in suitable animal hosts such as nude mice, and the like, invitro, in vivo, and ex vivo (Freshney, Culture of Animal Cells: A Manualof Basic Technique (3rd ed. 1994)).

The term “vector” means a polynucleotide capable of being duplicatedwithin a biological system or that can be moved between such systems.Vector polynucleotides typically contain elements, such as origins ofreplication, polyadenylation signal or selection markers that functionto facilitate the duplication or maintenance of these polynucleotides ina biological system. Examples of such biological systems may include acell, virus, animal, plant, and reconstituted biological systemsutilizing biological components capable of duplicating a vector. Thepolynucleotide comprising a vector may be DNA or RNA molecules or ahybrid of these.

The term “expression vector” means a vector that can be utilized in abiological system or in a reconstituted biological system to direct thetranslation of a polypeptide encoded by a polynucleotide sequencepresent in the expression vector.

The term “polynucleotide” means a molecule comprising a chain ofnucleotides covalently linked by a sugar-phosphate backbone or otherequivalent covalent chemistry. Double and single-stranded DNAs and RNAsare typical examples of polynucleotides.

The term “polypeptide” or “protein” means a molecule that comprises atleast two amino acid residues linked by a peptide bond to form apolypeptide. Small polypeptides of less than about 50 amino acids may bereferred to as “peptides”.

The term “bispecific EGFR/c-Met molecule” or “bispecific EGFR/c-Met FN3domain containing molecule” as used herein refers to a moleculecomprising an EGFR binding FN3 domain and a distinct c-Met binding FN3domain that are covalently linked together either directly or via alinker. An exemplary bispecific EGFR/c-Met binding molecule comprises afirst FN3 domain specifically binding EGFR and a second FN3 domainspecifically binding c-Met.

“Valent” as used herein refers to the presence of a specified number ofbinding sites specific for an antigen in a molecule. As such, the terms“monovalent”, “bivalent”, “tetravalent”, and “hexavalent” refer to thepresence of one, two, four and six binding sites, respectively, specificfor an antigen in a molecule.

“Mixture” as used herein refers to a sample or preparation of two ormore FN3 domains not covalently linked together. A mixture may consistof two or more identical FN3 domains or distinct FN3 domains.

Compositions of Matter

The present invention provides monospecific and bispecific EGFR and/orc-Met binding FN3 domain containing molecules. The present inventionprovides polynucleotides encoding the FN3 domains of the invention orcomplementary nucleic acids thereof, vectors, host cells, and methods ofmaking and using them.

Monospecific EGFR Binding Molecules

The present invention provides fibronectin type III (FN3) domains thatbind specifically to epidermal growth factor receptor (EGFR) and blockbinding of epidermal growth factor (EGF) to EGFR, and thus can be widelyused in therapeutic and diagnostic applications. The present inventionprovides polynucleotides encoding the FN3 domains of the invention orcomplementary nucleic acids thereof, vectors, host cells, and methods ofmaking and using them.

The FN3 domains of the invention bind EGFR with high affinity andinhibit EGFR signaling, and may provide a benefit in terms ofspecificity and reduced off-target toxicity when compared to smallmolecule EGFR inhibitors, and improved tissue penetration when comparedto conventional antibody therapeutics.

One embodiment of the invention an isolated fibronectin type III (FN3)domain that specifically binds epidermal growth factor receptor (EGFR)and blocks binding of epidermal growth factor (EGF) to EGFR.

The FN3 domains of the invention may block EGF binding to the EGFR withan IC₅₀ value of less than about 1×10⁻⁷ M, less than about 1×10⁻⁸ M,less than about 1×10⁻⁹ M, less than about 1×10⁻¹⁰ M, less than about1×10⁻¹¹ M, or less than about 1×10⁻¹² M in a competition assay employingA431 cells and detecting amount of fluorescence from bound biotinylatedEGF using streptavidin-phycoerythrin conjugate at 600 nM on A431 cellsincubated with or without the FN3 domains of the invention. ExemplaryFN3 domains may block EGF binding to the EGFR with an IC₅₀ value betweenabout 1×10⁻⁹ M to about 1×10⁻⁷ M, such as EGFR binding FN3 domainshaving the amino acid sequence of SEQ ID NOs: 18-29, 107-110, or122-137. The FN3 domains of the invention may block EGF binding to theEGFR by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% whencompared to binding of EGF to the EGFR in the absence of the FN3 domainsof the invention using the same assay conditions.

The FN3 domain of the invention may inhibit EGFR signaling by at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% when compared to thelevel of signaling in the absence of the FN3 domains of the inventionusing the same assay conditions.

Binding of a ligand such as EGF to EGFR stimulates receptordimerization, autophosphorylation, activation of the receptor'sinternal, cytoplasmic tyrosine kinase domain, and initiation of multiplesignal transduction and transactivation pathways involved in regulationof DNA synthesis (gene activation) and cell cycle progression ordivision. Inhibition of EGFR signaling may result in inhibition in oneor more EGFR downstream signaling pathways and therefore neutralizingEGFR may have various effects, including inhibition of cellproliferation and differentiation, angiogenesis, cell motility andmetastasis.

EGFR signaling may be measured using various well know methods, forexample measuring the autophosphorylation of the receptor at any of thetyrosines Y1068, Y1148, and Y1173 (Downward et al., Nature 311:483-5,1984) and/or phosphorylation of natural or synthetic substrates.Phosphorylation can be detected using well known methods such as anELISA assay or a western plot using a phosphotyrosine specific antibody.Exemplary assays can be found in Panek et al., J Pharmacol Exp Thera283:1433-44, 1997 and Batley et al., Life Sci 62:143-50, 1998, andassays described herein.

In one embodiment, the FN3 domain of the invention inhibits EGF-inducedEGFR phosphorylation at EGFR residue position Tyrosine 1173 with an IC₅₀value of less than about 2.5×10⁻⁶ M, for example less than about 1×10⁻⁶M, less than about 1×10⁻⁷ M, less than about 1×10⁻⁸ M, less than about1×10⁻⁹ M, less than about 1×10⁻¹⁰ M, less than about 1×10⁻¹¹ M, or lessthan about 1×10⁻¹² M when measured in A431 cells using 50 ng/mL humanEGF.

In one embodiment, the FN3 domain of the invention inhibits EGF-inducedEGFR phosphorylation at EGFR residue position Tyrosine 1173 with an IC₅₀value between about 1.8×10⁻⁸ M to about 2.5×10⁻⁶ M when measured in A431cells using 50 ng/mL human EGF. Such exemplary FN3 domains are thosehaving the amino acid sequence of SEQ ID NOs: 18-29, 107-110, or122-137.

In one embodiment, the FN3 domain of the invention binds human EGFR witha dissociation constant (K_(D)) of less than about 1×10⁻⁸ M, for exampleless than about 1×10⁻⁹ M, less than about 1×10⁻¹⁰ M, less than about1×10⁻¹¹ M, less than about 1×10⁻¹² M, or less than about 1×10⁻¹³ M asdetermined by surface plasmon resonance or the Kinexa method, aspracticed by those of skill in the art. In some embodiments, the FN3domain of the invention binds human EGFR with a K_(D) of between about2×10⁻¹⁰ to about 1×10⁻⁸ M. The affinity of a FN3 domain for EGFR can bedetermined experimentally using any suitable method. (See, for example,Berzofsky, et al., “Antibody-Antigen Interactions,” In FundamentalImmunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby,Janis Immunology, W. H. Freeman and Company: New York, N.Y. (1992); andmethods described herein). The measured affinity of a particular FN3domain-antigen interaction can vary if measured under differentconditions (e.g., osmolarity, pH). Thus, measurements of affinity andother antigen-binding parameters (e.g., K_(D), K_(on), K_(off)) arepreferably made with standardized solutions of protein scaffold andantigen, and a standardized buffer, such as the buffer described herein.

Exemplary FN3 domains of the invention that bind EGFR include FN3domains of SEQ ID NOs: 18-29, 107-110, or 122-137.

In one embodiment, the FN3 domain that specifically binds EGFR comprisesan amino acid sequence at least 87% identical to the amino acid sequenceof SEQ ID NO: 27.

In one embodiment, the FN3 domain that specifically binds EGFR comprises

an FG loop comprising the sequence HNVYKDTNX₉RGL (SEQ ID NO: 179) or thesequence LGSYVFEHDVML (SEQ ID NO: 180), wherein X₉ is M or I; and

a BC loop comprising the sequence X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO: 181),

wherein

-   -   X₁ is A, T, G or D;    -   X₂ is A, D, Y or W;    -   X₃ is P, D or N;    -   X₄ is L or absent;    -   X₅ is D, H, R, G, Y or W;    -   X₆ is G, D or A;    -   X₇ is A, F, G, H or D; and    -   X₈ is Y, F or L.

The FN3 domains of the invention that specifically bind EGFR and inhibitautophosphorylation of EGFR may comprise as a structural feature an FGloop comprising the sequence HNVYKDTNX₉RGL (SEQ ID NO: 179) or thesequence LGSYVFEHDVML (SEQ ID NO: 180), wherein X₉ is M or I. Such FN3domains may further comprise a BC loop of 8 or 9 amino acids in lengthand defined by the sequence X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO: 181), andinhibit EGFR autophosphorylation with an IC₅₀ value of less than about2.5×10⁻⁶ M, or with an IC₅₀ value of between about 1.8×10⁻⁸ M to about2.5×10⁻⁶ M when measured in A431 cells using 50 ng/mL human EGF.

The FN3 domains of the invention that specifically bind EGFR and inhibitautophosphorylation of EGFR further comprise the sequence of

LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNX₉RGLPLSAEFTT (SEQ ID NO: 182), or thesequence

LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVLGSYVFEHDVMLPLSAEFTT (SEQ ID NO: 183),

wherein

-   -   X₁ is A, T, G or D;    -   X₂ is A, D, Y or W;    -   X₃ is P, D or N;    -   X₄ is L or absent;    -   X₅ is D, H, R, G, Y or W;    -   X₆ is G, D or A;    -   X₇ is A, F, G, H or D;    -   X₈ is Y, F or L; and    -   X₉ is M or I

The EGFR binding FN3 domains can be generated and tested for theirability to inhibit EGFR autophosphorylation using well known methods andmethods described herein.

Another embodiment of the invention is an isolated FN3 domain thatspecifically binds EGFR, wherein the FN3 domain comprises the sequenceshown in SEQ ID NOs: 18-29, 107-110, 122-137 or 194-211.

In some embodiments, the EGFR binding FN3 domains comprise an initiatormethionine (Met) linked to the N-terminus or a cysteine (Cys) linked toa C-terminus of a particular FN3 domain, for example to facilitateexpression and/or conjugation of half-life extending molecules.

Another embodiment of the invention is an isolated fibronectin type III(FN3) domain that specifically binds EGFR and blocks binding of EGF tothe EGFR, wherein the FN3 domain is isolated from a library designedbased on Tencon sequence of SEQ ID NO: 1.

Another embodiment of the invention is an isolated fibronectin type III(FN3) domain that specifically binds EGFR and blocks binding of EGF tothe EGFR, wherein the FN3 domain binds EGFR with one or more amino acidresidues corresponding to residues D23, F27, Y28, V77 and G85 ofP54AR4-83v2 (SEQ ID NO: 27).

Amino acid residues contributing to FN3 domain binding to EGFR can beidentified using methods such as mutagenesis and evaluating of bindingresidues/surface by crystal structure. Substitutions at residues D23,F27, Y28, V77, G85 in EGFR binding FN3 domain P54AR4-83v2 (SEQ ID NO:27) reduced EGFR binding to the FN3 domain by greater than 100-fold.EGFR-binding FN3 domains P54AR4-48, P54AR4-81, P53A1R5-17v2,P54AR4-83v22 and P54AR4-83v23 share these residues and can be expectedto bind to EGFR with the same paratope residues as P54AR4-83v2. OtherEGFR binding FN3 domains can be created by holding positions D23, F27,Y28, V77, G85 constant while changing the amino acids located at theother positions of the BC and FG loops (positions 24, 25, 75, 76, 78,79, 80, 81, 82, 83, 84, and 86). These changes can be done by design ofspecific amino acids at specific positions or by incorporation of thesepositions into a library that replaces these sites with random aminoacids. New FN3 domains designed in such a way can be used to screen foror select for optimized properties such as EGFR binding, solubility,stability, immunogenicity or serum half-life.

Monospecific c-Met Binding Molecules

The present invention provides fibronectin type III (FN3) domains thatbind specifically to hepatocyte growth factor receptor (c-Met) and blockbinding of hepatocyte growth factor (HGF) to c-Met, and thus can bewidely used in therapeutic and diagnostic applications. The presentinvention provides polynucleotides encoding the FN3 domains of theinvention or complementary nucleic acids thereof, vectors, host cells,and methods of making and using them.

The FN3 domains of the invention bind c-Met with high affinity andinhibit c-Met signaling, and may provide a benefit in terms ofspecificity and reduced off-target toxicity when compared to smallmolecule c-Met inhibitors, and improved tissue penetration when comparedto conventional antibody therapeutics. The FN3 domains of the inventionare monovalent, therefore preventing unwanted receptor clustering andactivation that may occur with other bivalent molecules.

One embodiment of the invention an isolated fibronectin type III (FN3)domain that specifically binds hepatocyte growth factor receptor (c-Met)and blocks binding of hepatocyte growth factor (HGF) to c-Met.

The FN3 domains of the invention may block HGF binding to c-Met with anIC₅₀ value of less than about 1×10⁻⁷ M, less than about 1×10⁻⁸ M, lessthan about 1×10⁻⁹ M, less than about 1×10⁻¹⁰ M, less than about 1×10⁻¹¹M, or less than about 1×10⁻¹² M in an assay detecting inhibition ofbinding of biotinylated HGF to c-Met-Fc fusion protein in the presenceof the FN3 domains of the invention. Exemplary FN3 domains my block HGFbinding to the c-Met with an IC₅₀ value between about 2×10⁻¹⁰ M to about6×10⁻⁸M. The FN3 domains of the invention may block HGF binding to c-Metby at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% when comparedto binding of HGF to c-Met in the absence of the FN3 domains of theinvention using the same assay conditions.

The FN3 domain of the invention may inhibit c-Met signaling by at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% when compared to thelevel of signaling in the absence of FN3 domains of the invention usingthe same assay conditions.

Binding of HGF to c-Met stimulates receptor dimerization,autophosphorylation, activation of the receptor's internal, cytoplasmictyrosine kinase domain, and initiation of multiple signal transductionand transactivation pathways involved in regulation of DNA synthesis(gene activation) and cell cycle progression or division. Inhibition ofc-Met signaling may result in inhibition of one or more c-Met downstreamsignaling pathways and therefore neutralizing c-Met may have variouseffects, including inhibition of cell proliferation and differentiation,angiogenesis, cell motility and metastasis.

c-Met signaling may be measured using various well know methods, forexample measuring the autophosphorylation of the receptor on at leastone tyrosine residues Y1230, Y1234, Y1235 or Y1349, and/orphosphorylation of natural or synthetic substrates. Phosphorylation canbe detected, for example, using an antibody specific for phosphotyrosinein an ELISA assay or on a western blot. Assays for tyrosine kinaseactivity (Panek et al., J Pharmacol Exp Thera 283:1433-44, 1997; Batleyet al., Life Sci 62:143-50, 1998), and assays described herein.

In one embodiment, the FN3 domain of the invention inhibits HGF-inducedc-Met phosphorylation at c-Met residue position 1349 with an IC₅₀ valueof less than about 1×10⁻⁶ M, less than about 1×10⁻¹¹ M, less than about1×10⁻⁸ M, less than about 1×10⁻⁹ M, less than about 1×10⁻¹⁰ M, less thanabout 1×10⁻¹¹ M, or less than about 1×10⁻¹² M when measured in NCI-H441cells using 100 ng/mL recombinant human HGF.

In one embodiment, the FN3 domain of the invention inhibits HGF-inducedc-Met phosphorylation at c-Met tyrosine Y1349 with an IC₅₀ value betweenabout 4×10⁻⁹ M to about 1×10⁻⁶ M when measured in NCI-H441 cells using100 ng/mL recombinant human HGF.

In one embodiment, the FN3 domain of the invention binds human c-Metwith an dissociation constant (K_(D)) of equal to or less than about1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹M, 1×10⁻¹² M, 1×10⁻¹³ M,1×10⁻¹⁴ M, or 1×10⁻⁵M as determined by surface plasmon resonance or theKinexa method, as practiced by those of skill in the art. In someembodiments, the FN3 domain of the invention binds human c-Met with aK_(D) of between about 3×10⁻¹⁰ M to about 5×10⁻⁸ M. The affinity of aFN3 domain for c-Met can be determined experimentally using any suitablemethod. (See, for example, Berzofsky, et al., “Antibody-AntigenInteractions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press:New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman andCompany: New York, N.Y. (1992); and methods described herein). Themeasured affinity of a particular FN3 domain-antigen interaction canvary if measured under different conditions (e.g., osmolarity, pH).Thus, measurements of affinity and other antigen-binding parameters(e.g., K_(D), K_(on), K_(off)) are preferably made with standardizedsolutions of protein scaffold and antigen, and a standardized buffer,such as the buffer described herein.

Exemplary FN3 domains of the invention that bind c-Met include FN3domains having the amino acid sequence of SEQ ID NOs: 32-49, 111-114 or212-223.

In one embodiment, the FN3 domain that specifically binds c-Metcomprises an amino acid sequence at least 83% identical to the aminoacid sequence of SEQ ID NO: 41.

In one embodiment, the FN3 domain that specifically binds c-Metcomprises

-   -   a C strand and a CD loop comprising the sequence DSFX₁₀IRYX₁₁E    -   X₁₂X₁₃X₁₄X₁₅GX₁₆ (SEQ ID NO: 184), wherein        -   X₁₀ is W, F or V;        -   X₁₁ is D, F or L;        -   X₁₂ is V, F or L;        -   X₁₃ is V, L or T;        -   X₁₄ is V, R, G, L, T or S;        -   X₁₅ is G, S, A, T or K; and        -   X₁₆ is E or D; and    -   a F strand and a FG loop comprising the sequence        TEYX₁₇VX₁₈IX₁₉X₂₀V KGGX₂₁X₂₂SX₂₃ (SEQ ID NO: 185), wherein        -   X₁₇ is Y, W, I, V, G or A;        -   X₁₈ is N, T, Q or G;        -   X₁₉ is L, M, N or I;        -   X₂₀ is G or S;        -   X₂₁ is S, L, G, Y, T, R, H or K;        -   X₂₂ is I, V or L; and        -   X₂₃ is V, T, H, I, P, Y or L.

The FN3 domains of the invention that specifically bind c-Met andinhibit autophosphorylation of c-Met further comprises the sequence:

LPAPKNLVVSRVTEDSARLSWTAPDAAF DSFX₁₀IRYX₁₁E X₁₂X₁₃X₁₄X₁₅GX₁₆AIVLTVPGSERSYDLTGLKPGTEYX₁₇VX₁₈IX₁₉X₂₀VKGGX₂₁X₂₂SX₂₃PLSAEFTT (SEQ ID NO:186),

wherein

-   -   X₁₀ is W, F or V; and    -   X₁₁ is D, F or L;    -   X₁₂ is V, F or L;    -   X₁₃ is V, L or T;    -   X₁₄ is V, R, G, L, T or S;    -   X₁₅ is G, S, A, T or K;    -   X₁₆ is E or D;    -   X₁₇ is Y, W, I, V, G or A;    -   X₁₈ is N, T, Q or G;    -   X₁₉ is L, M, N or I;    -   X₂₀ is G or S;    -   X₂₁ is S, L, G, Y, T, R, H or K;    -   X₂₂ is I, V or L; and    -   X₂₃ is V, T, H, I, P, Y or L.

Another embodiment of the invention is an isolated FN3 domain thatspecifically binds c-Met, wherein the FN3 domain comprises the sequenceshown in SEQ ID NOs: 32-49 or 111-114.

Another embodiment of the invention is an isolated fibronectin type III(FN3) domain that specifically binds c-Met and blocks binding of HGF tothe c-Met, wherein the FN3 domain is isolated from a library designedbased on Tencon sequence of SEQ ID NO: 1.

Another embodiment of the invention is an isolated fibronectin type III(FN3) domain that specifically binds c-Met and blocks binding of HGF toc-Met, wherein the FN3 domain binds c-Met with one or more amino acidresidues corresponding to residues R34, F38, M72 and 179 inP114AR7P95-A3 (SEQ ID NO: 41).

Amino acid residues contributing to FN3 domain binding to c-Met can beidentified using methods such as mutagenesis and evaluating of bindingresidues/surface by crystal structure. Substitutions at residues R34S,F38S, M72S and 1795 in the c-Met-binding FN3 domain P114AR7P95-A3 (SEQID NO: 27) reduced c-Met binding to the FN3 domain by greater than100-fold. c-Met-binding FN3 domains molecules P114AR7P92-F3,P114AR7P95-D3, P114AR7P95-F10 and P114AR7P95-H8 share these residues andcan be expected to bind to c-Met with the same paratope residues asP114AR7P95-A3. Other c-Met binding FN3 domains can be created by holdingpositions R34S, F38S, M72S and I79S constant while changing the aminoacids located at the other positions of the C-strand, F-strand, CD-Loopand/or FG-loops (positions 32, 36, 39, 40, 68, 70, 78, and 81). Thesechanges can be done by design of specific amino acids at specificpositions or by incorporation of these positions into a library thatreplaces these sites with random amino acids. New FN3 domains designedin such a way can be used to screen for or select for optimizedproperties such as c-Met binding, solubility, stability, immunogenicity,or serum half-life.

Isolation of EGFR or c-Met Binding FN3 Domains from a Library Based onTencon Sequence

Tencon (SEQ ID NO: 1) is a non-naturally occurring fibronectin type III(FN3) domain designed from a consensus sequence of fifteen FN3 domainsfrom human tenascin-C (Jacobs et al., Protein Engineering, Design, andSelection, 25:107-117, 2012; U.S. Pat. Publ. No. 2010/0216708). Thecrystal structure of Tencon shows six surface-exposed loops that connectseven beta-strands as is characteristic to the FN3 domains, thebeta-strands referred to as A, B, C, D, E, F, and G, and the loopsreferred to as AB, BC, CD, DE, EF, and FG loops (Bork and Doolittle,Proc Natl Acad Sci USA 89:8990-8992, 1992; U.S. Pat. No. 6,673,901).These loops, or selected residues within each loop, can be randomized inorder to construct libraries of fibronectin type III (FN3) domains thatcan be used to select novel molecules that bind EGFR or c-Met. Table 1shows positions and sequences of each loop and beta-strand in Tencon(SEQ ID NO: 1).

Library designed based on Tencon sequence may thus have randomized FGloop, or randomized BC and FG loops, such as libraries TCL1 or TCL2 asdescribed below. The Tencon BC loop is 7 amino acids long, thus 1, 2, 3,4, 5, 6 or 7 amino acids may be randomized in the library diversified atthe BC loop and designed based on Tencon sequence. The Tencon FG loop is7 amino acids long, thus 1, 2, 3, 4, 5, 6 or 7 amino acids may berandomized in the library diversified at the FG loop and designed basedon Tencon sequence. Further diversity at loops in the Tencon librariesmay be achieved by insertion and/or deletions of residues at loops. Forexample, the FG and/or BC loops may be extended by 1-22 amino acids, ordecreased by 1-3 amino acids. The FG loop in Tencon is 7 amino acidslong, whereas the corresponding loop in antibody heavy chains rangesfrom 4-28 residues. To provide maximum diversity, the FG loop may bediversified in sequence as well as in length to correspond to theantibody CDR3 length range of 4-28 residues. For example, the FG loopcan further be diversified in length by extending the loop by additional1, 2, 3, 4 or 5 amino acids.

Library designed based on Tencon sequence may also have randomizedalternative surfaces that form on a side of the FN3 domain and comprisetwo or more beta strands, and at least one loop. One such alternativesurface is formed by amino acids in the C and the F beta-strands and theCD and the FG loops (a C-CD-F-FG surface). A library design based onTencon alternative C-CD-F-FG surface and is shown in FIG. 1 and detailedgeneration of such libraries is described in U.S. Pat. Publ. No.US2013/0226834. Library designed based on Tencon sequence also includeslibraries designed based on Tencon variants, such as Tencon variantshaving substitutions at residues positions 11, 14, 17, 37, 46, 73, or 86(residue numbering corresponding to SEQ ID NO: 1), and which variantsdisplay improve thermal stability. Exemplary Tencon variants aredescribed in US Pat. Publ. No. 2011/0274623, and include Tencon27 (SEQID NO: 99) having substitutions E11R, L17A, N46V and E86I when comparedto Tencon of SEQ ID NO: 1.

TABLE 1 Tencon FN3 domain (SEQ ID NO: 1) A strand  1-12 AB loop 13-16 Bstrand 17-21 BC loop 22-28 C strand 29-37 CD loop 38-43 D strand 44-50DE loop 51-54 E strand 55-59 EF loop 60-64 F strand 65-74 FG loop 75-81G strand 82-89

Tencon and other FN3 sequence based libraries can be randomized atchosen residue positions using a random or defined set of amino acids.For example, variants in the library having random substitutions can begenerated using NNK codons, which encode all 20 naturally occurringamino acids. In other diversification schemes, DVK codons can be used toencode amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp,Glu, Gly, and Cys. Alternatively, NNS codons can be used to give rise toall 20 amino acid residues and simultaneously reducing the frequency ofstop codons. Libraries of FN3 domains with biased amino aciddistribution at positions to be diversified can be synthesized forexample using Slonomics® technology (http:_//www_sloning_com). Thistechnology uses a library of pre-made double stranded triplets that actas universal building blocks sufficient for thousands of gene synthesisprocesses. The triplet library represents all possible sequencecombinations necessary to build any desired DNA molecule. The codondesignations are according to the well known IUB code.

The FN3 domains specifically binding EGFR or c-Met of the invention canbe isolated by producing the FN3 library such as the Tencon libraryusing cis display to ligate DNA fragments encoding the scaffold proteinsto a DNA fragment encoding RepA to generate a pool of protein-DNAcomplexes formed after in vitro translation wherein each protein isstably associated with the DNA that encodes it (U.S. Pat. No. 7,842,476;Odegrip et al., Proc Natl Acad Sci USA 101, 2806-2810, 2004), andassaying the library for specific binding to EGFR and/or c-Met by anymethod known in the art and described in the Example Exemplary wellknown methods which can be used are ELISA, sandwich immunoassays, andcompetitive and non-competitive assays (see, e.g., Ausubel et al., eds,1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,Inc., New York). The identified FN3 domains specifically binding EGFR orc-Met are further characterized for their ability to block EGFR ligandsuch as EGF binding to EGFR, or HGF binding to c-Met, and for theirability to inhibit EGFR and/or c-Met signaling using methods describedherein.

The FN3 domains specifically binding to EGFR or c-Met of the inventioncan be generated using any FN3 domain as a template to generate alibrary and screening the library for molecules specifically bindingEGFR or c-Met using methods provided within. Exemplar FN3 domains thatcan be used are the 3rd FN3 domain of tenascin C (TN3) (SEQ ID NO: 75),Fibcon (SEQ ID NO: 76), and the 10^(th) FN3 domain of fibronectin (FN10)(SEQ ID NO: 77). Standard cloning and expression techniques are used toclone the libraries into a vector or synthesize double stranded cDNAcassettes of the library, to express, or to translate the libraries invitro. For example ribosome display (Hanes and Pluckthun, Proc Natl AcadSci USA, 94, 4937-4942, 1997), mRNA display (Roberts and Szostak, ProcNatl Acad Sci USA, 94, 12297-12302, 1997), or other cell-free systems(U.S. Pat. No. 5,643,768) can be used. The libraries of the FN3 domainvariants may be expressed as fusion proteins displayed on the surfacefor example of any suitable bacteriophage. Methods for displaying fusionpolypeptides on the surface of a bacteriophage are well known (U.S. Pat.Publ. No. 2011/0118144; Int. Pat. Publ. No. WO2009/085462; U.S. Pat.Nos. 6,969,108; 6,172,197; 5,223,409; 6,582,915; 6,472,147).

The FN3 domains specifically binding EGFR or c-Met of the invention canbe modified to improve their properties such as improve thermalstability and reversibility of thermal folding and unfolding. Severalmethods have been applied to increase the apparent thermal stability ofproteins and enzymes, including rational design based on comparison tohighly similar thermostable sequences, design of stabilizing disulfidebridges, mutations to increase alpha-helix propensity, engineering ofsalt bridges, alteration of the surface charge of the protein, directedevolution, and composition of consensus sequences (Lehmann and Wyss,Curr Opin Biotechnol, 12, 371-375, 2001). High thermal stability mayincrease the yield of the expressed protein, improve solubility oractivity, decrease immunogenicity, and minimize the need of a cold chainin manufacturing. Residues that can be substituted to improve thermalstability of Tencon (SEQ ID NO: 1) are residue positions 11, 14, 17, 37,46, 73, or 86, and are described in US Pat. Publ. No. 2011/0274623.Substitutions corresponding to these residues can be incorporated to theFN3 domains or the bispecific FN3 domain containing molecules of theinvention.

Another embodiment of the invention is an isolated FN3 domain thatspecifically binds EGFR and blocks binding of EGF to EGFR, comprisingthe sequence shown in SEQ ID NOs: 18-29, 107-110, 122-137, furthercomprising substitutions at one or more residue positions correspondingto positions 11, 14, 17, 37, 46, 73, and 86 in Tencon (SEQ ID NO: 1).

Another embodiment of the invention is an isolated FN3 domain thatspecifically binds c-Met and blocks binding of HGF to c-Met, comprisingthe sequence shown in SEQ ID NOs: 32-49 or 111-114, further comprisingsubstitutions at one or more residue positions corresponding topositions 11, 14, 17, 37, 46, 73, and 86 in Tencon (SEQ ID NO: 1).

Exemplary substitutions are substitutions E11N, E14P, L17A, E37P, N46V,G73Y and E86I (numbering according to SEQ ID NO: 1).

In some embodiments, the FN3 domains of the invention comprisesubstitutions corresponding to substitutions L17A, N46V, and E86I inTencon (SEQ ID NO: 1).

The FN3 domains specifically binding EGFR (FIG. 1) have an extended FGloop when compared to Tencon (SEQ ID NO: 1). Therefore, the residuescorresponding to residues 11, 14, 17, 37, 46, 73, and 86 in Tencon (SEQID NO: 1) are residues 11, 14, 17, 37, 46, 73 and 91 in EGFR FN3 domainsshown in FIGS. 1A and 1B except for the FN3 domain of SEQ ID NO: 24,wherein the corresponding residues are residues 11, 14, 17, 38, 74, and92 due to an insertion of one amino acid in the BC loop.

Another embodiment of the invention is an isolated FN3 domain thatspecifically binds EGFR and blocks binding of EGF to EGFR comprising theamino acid sequence shown in SEQ ID NOs: 18-29, 107-110, 122-137 or194-211, optionally having one, two or three substitutions correspondingto substitutions L17A, N46V and E86I in Tencon (SEQ ID NO: 1).

Another embodiment of the invention is an isolated FN3 domain thatspecifically binds c-Met and blocks binding of HGF to c-Met comprisingthe amino acid sequence shown in SEQ ID NOs: 32-49, 111-114 or 212-223,optionally having one, two or three substitutions corresponding tosubstitutions L17A, N46V, and E86I in Tencon (SEQ ID NO: 1).

Measurement of protein stability and protein lability can be viewed asthe same or different aspects of protein integrity. Proteins aresensitive or “labile” to denaturation caused by heat, by ultraviolet orionizing radiation, changes in the ambient osmolarity and pH if inliquid solution, mechanical shear force imposed by small pore-sizefiltration, ultraviolet radiation, ionizing radiation, such as by gammairradiation, chemical or heat dehydration, or any other action or forcethat may cause protein structure disruption. The stability of themolecule can be determined using standard methods. For example, thestability of a molecule can be determined by measuring the thermalmelting (“TM”) temperature, the temperature in ° Celsius (° C.) at whichhalf of the molecules become unfolded, using standard methods.Typically, the higher the TM, the more stable the molecule. In additionto heat, the chemical environment also changes the ability of theprotein to maintain a particular three dimensional structure.

In one embodiment, the FN3 domains binding EGFR or c-Met of theinvention exhibit increased stability by at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or95% or more compared to the same domain prior to engineering measured bythe increase in the TM.

Chemical denaturation can likewise be measured by a variety of methods.Chemical denaturants include guanidinium hydrochloride, guanidiniumthiocyanate, urea, acetone, organic solvents (DMF, benzene,acetonitrile), salts (ammonium sulfate, lithium bromide, lithiumchloride, sodium bromide, calcium chloride, sodium chloride); reducingagents (e.g. dithiothreitol, beta-mercaptoethanol, dinitrothiobenzene,and hydrides, such as sodium borohydride), non-ionic and ionicdetergents, acids (e.g. hydrochloric acid (HCl), acetic acid (CH₃COOH),halogenated acetic acids), hydrophobic molecules (e.g. phosopholipids),and targeted denaturants. Quantitation of the extent of denaturation canrely on loss of a functional property, such as ability to bind a targetmolecule, or by physiochemical properties, such as tendency toaggregation, exposure of formerly solvent inaccessible residues, ordisruption or formation of disulfide bonds.

In one embodiment, the FN3 domain of the invention binding EGFR or c-Metexhibit increased stability by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% ormore compared to the same scaffold prior to engineering, measured byusing guanidinium hydrochloride as a chemical denaturant. Increasedstability can be measured as a function of decreased tryptophanfluorescence upon treatment with increasing concentrations of guanidinehydrochloride using well known methods.

The FN3 domains of the invention may be generated as monomers, dimers,or multimers, for example, as a means to increase the valency and thusthe avidity of target molecule binding, or to generate bi- ormultispecific scaffolds simultaneously binding two or more differenttarget molecules. The dimers and multimers may be generated by linkingmonospecific, bi- or multispecific protein scaffolds, for example, bythe inclusion of an amino acid linker, for example a linker containingpoly-glycine, glycine and serine, or alanine and proline. Exemplarylinker include (GS)₂, (SEQ ID NO: 78), (GGGS)₂ (SEQ ID NO: 224),(GGGGS)₅ (SEQ ID NO: 79), (AP)₂ (SEQ ID NO: 80), (AP)₅ (SEQ ID NO: 81),(AP)₁₀ (SEQ ID NO: 82), (AP)₂₀ (SEQ ID NO: 83) and A(EAAAK)₅AAA (SEQ IDNO: 84). The dimers and multimers may be linked to each other in a N- toC-direction. The use of naturally occurring as well as artificialpeptide linkers to connect polypeptides into novel linked fusionpolypeptides is well known in the literature (Hallewell et al., J BiolChem 264, 5260-5268, 1989; Alfthan et al., Protein Eng. 8, 725-731,1995; Robinson & Sauer, Biochemistry 35, 109-116, 1996; U.S. Pat. No.5,856,456).

Bispecific EGFR/c/Met Binding Molecules

The bispecific EGFR/c-Met FN3 domain containing molecules of theinvention may provide a benefit in terms of specificity and reducedoff-target toxicity when compared to small molecule EGFR inhibitors, andimproved tissue penetration when compared to conventional antibodytherapeutics. The present invention is based at least in part on thesurprising finding that the bispecific EGFR/c-Met FN3 domain containingmolecules of the invention provide a significantly improved synergisticinhibitory effect when compared to a mixture of EGFR-binding andc-Met-binding FN3 domains. The molecules may be tailored to specificaffinity towards both EGFR and c-Met to maximize tumor penetration andretention. The bispecific EGFR/c-Met FN3 domain containing molecules ofthe invention provide more efficient inhibition of EGFR and/or c-Metsignaling pathways and inhibit tumor growth more efficiently thancetuximab (Eribtux®)

One embodiment of the invention is an isolated bispecific FN3 domaincontaining molecule comprising a first fibronectin type III (FN3) domainand a second FN3 domain, wherein the first FN3 domain specifically bindsepidermal growth factor receptor (EGFR) and blocks binding of epidermalgrowth factor (EGF) to EGFR, and the second FN3 domain specificallybinds hepatocyte growth factor receptor (c-Met) and blocks binding ofhepatocyte growth factor (HGF) to c-Met.

The bispecific EGFR/c-Met FN3 domain containing molecules of theinvention can be generated by covalently linking any EGFR-binding FN3domain and any c-Met-binding FN3 domain of the invention directly or viaa linker. Therefore, the first FN3 domain of the bispecific molecule mayhave characteristics as described above for the EGFR-binding FN3domains, and the second FN3 domain of the bispecific molecule may havecharacteristics as described above for the c-Met-binding FN3 domains.

In one embodiment, the first FN3 domain of the bispecific EGFR/c-Met FN3domain containing molecule inhibits EGF-induced EGFR phosphorylation atEGFR residue Tyrosine 1173 with an IC₅₀ value of less than about2.5×10⁻⁶ M when measured in A431 cells using 50 ng/mL human EGF, and thesecond FN3 domain of the bispecific EGFR/c-Met FN3 domain containingmolecule inhibits HGF-induced c-Met phosphorylation at c-Met residueTyrosine 1349 with an IC₅₀ value of less than about 1.5×10⁻⁶ M whenmeasured in NCI-H441 cells using 100 ng/mL human HGF.

In another embodiment, the first FN3 domain of the bispecific EGFR/c-MetFN3 domain containing molecule inhibits EGF-induced EGFR phosphorylationat EGFR residue Tyrosine 1173 with an IC₅₀ value of between about1.8×10⁻⁸ M to about 2.5×10⁻⁶ M when measured in NCI-H292 cells using 50ng/mL human EGF, and the second FN3 domain of the bispecific EGFR/c-MetFN3 domain containing molecule inhibits HGF-induced c-Metphosphorylation at c-Met residue Tyrosine 1349 with an IC₅₀ valuebetween about 4×10⁻⁹ M to about 1.5×10⁻⁶ M when measured in NCI-H441cells using 100 ng/mL human HGF.

In another embodiment, the first FN3 domain of the bispecific EGFR/c-MetFN3 domain containing molecule binds human EGFR with a dissociationconstant (K_(D)) of less than about 1×10⁻⁸ M, and the second FN3 domainof the bispecific EGFR/c-Met FN3 domain containing molecule binds humanc-Met with a K_(D) of less than about 5×10⁻⁸ M.

In the bispecific molecule binding both EGFR and c-Met, the first FN3domain binds human EGFR with a K_(D) of between about 2×10⁻¹⁰ to about1×10⁻⁸ M, and the second FN3 domain binds human c-Met with a K_(D) ofbetween about 3×10⁻¹⁰ to about 5×10⁻⁸ M.

The affinity of the bispecific EGFR/c-Met molecule for EGFR and c-Metcan be determined as described in Example 3 and Example 5 for themonospecific molecules.

The first FN3 domain in the bispecific EGFR/c-Met molecule of theinvention may block EGF binding to EGFR with an IC₅₀ value of betweenabout 1×10⁻⁹ M to about 1.5×10⁻⁷ M in an assay employing A431 cells anddetecting the amount of fluorescence from bound biotinylated EGF usingstreptavidin-phycoerythrin conjugate at 600 nM on A431 cells incubatedwith or without the first FN3 domain. The first FN3 domain in thebispecific EGFR/c-Met molecule of the invention may block EGF binding tothe EGFR by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% whencompared to binding of EGF to EGFR in the absence of the first FN3domains using the same assay conditions.

The second FN3 domain in the bispecific EGFR/c-Met molecule of theinvention may block HGF binding to c-Met with an IC₅₀ value of betweenabout 2×10⁻¹⁰ M to about 6×10⁻⁸ M in an assay detecting inhibition ofbinding of biotinylated HGF to c-Met-Fc fusion protein in the presenceof the second FN3 domain. The second FN3 domain in the bispecificEGFR/c-Met molecule may block HGF binding to c-Met by at least 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% when compared to binding of HGF toc-Met in the absence of the second FN3 domain using the same assayconditions.

The bispecific EGFR/c-Met molecule of the invention may inhibit EGFRand/or c-Met signaling by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% when compared to the level of signaling in the absence ofthe bispecific EGFR/c-Met molecule of the invention using the same assayconditions.

EGFR and c-Met signaling may be measured using various well know methodsas described above for the monospecific molecules.

The bispecific EGFR/c-Met molecules of the invention comprising thefirst FN3 domain specifically binding EGFR and the second FN3 domainspecifically binding c-Met provide a significantly increased synergisticinhibition of EGFR and c/Met signaling and tumor cell proliferation whencompared to the synergistic inhibition observed by a mixture of thefirst and the second FN3 domain. Synergistic inhibition can be assessedfor example by measuring inhibition of ERK phosphorylation by thebispecific EGFR/c-Met FN3 domain containing molecules and by a mixtureof two monospecific molecules, one binding EGFR and the other c-Met. Thebispecific EGFR/c-Met molecules of the invention may inhibit ERKphosphorylation with an at least about 100 fold smaller, for example atleast 500, 1000, 5000 or 10,000 fold smaller IC₅₀ value when compared tothe IC₅₀ value for a mixture of two monospecific FN3 domains, indicatingat least 100 fold increased potency for the bispecific EGFR/c-Met FN3domain containing molecules when compared to the mixture of twomonospecific FN3 domains. Exemplary bispecific EGFR-c-Met FN3 domaincontaining molecules may inhibit ERK phosphorylation with and IC₅₀ valueof about 5×10⁻⁹ M or less. ERK phosphorylation can be measured usingstandard methods and methods described herein.

The bispecific EGFR/c-Met FN3 domain containing molecule of theinvention may inhibit H292 cell proliferation with an IC₅₀ value that isat least 30-fold less when compared to the IC₅₀ value of inhibition ofH292 cell growth with a mixture of the first FN3 domain and the secondFN3, wherein the cell proliferation is induced with medium containing10% FBS supplemented with 7.5 ng/mL HGF. The bispecific molecule of theinvention may inhibit tumor cell proliferation with an IC₅₀ value thatis about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600,700, 800, or about 1000 fold less when compared to the IC₅₀ value ofinhibition of tumor cell proliferation with a mixture of the first FN3domain and the second FN3 domain. Inhibition of tumor cell proliferationcan be measured using standard methods and methods described herein.

In some embodiments, the bispecific EGFR/c-Met FN3 domain containingmolecule binds EGFR with one or more amino acid residues correspondingto residues D23, F27, Y28, V77 and G85 of P54AR4-83v2 (SEQ ID NO: 27).

In other embodiments, the bispecific EGFR/c-Met FN3 domain containingmolecule binds c-Met with one or more amino acid residues correspondingto residues R34, F38, M72 and 179 in P114AR7P95-A3 (SEQ ID NO: 41).

Paratope residues in the bispecific molecules can be identified bymutagenesis studies or from co-crystal structures of the FN3 domain andEGFR or c-Met. Mutagenesis studies may be employed by for example usingalanine scanning, and the resulting variants may be tested for theirbinding to EGFR or c-Met using standard methods. Typically paratoperesidues are those residues that when mutagenized, result in variantswith reduced or abolished binding to EGFR or c-Met. EGFR-binding FN3domains with substitutions at amino acid residue positions correspondingto residues D23, F27, Y28, V77 and G85 of P54AR4-83v2 (SEQ ID NO: 27),when substituted, reduce EGFR binding at least 100-fold when compared tothe wild type P54AR4-83v2. Bispecific molecules ECB1, ECB2, ECB3, ECB4,ECB5, ECB6, ECB7, ECB15, ECB17, ECB60, ECB37, ECB94, ECB95, ECB96,ECB97, ECB91, ECB18, ECB28, ECB38, ECB39, ECB168 and ECB176 have D, F,Y, V and G at residue positions corresponding to residues D23, F27, Y28,V77 and G85 of P54AR4-83v2 and expected to bind to EGFR with theseresidues. c-Met binding FN3 domains with substitutions at amino acidresidue positions corresponding to residues R34, F38, M72 and 179 ofP114AR7P95-A3 (SEQ ID NO: 41), when substituted, abolish or reduce c-Metbinding at least 100-fold when compared to the wild type P114AR7P95-A3.Bispecific molecules ECB2, ECB5, ECB15, ECB60, ECB38 and ECB39 have R,F, M and I at residue positions corresponding to residues R34, F38, M72and 179 of P114AR7P95-A3 (SEQ ID NO: 41) and expected to bind to c-Metwith these residues.

Another embodiment of the invention is a bispecific FN3 domaincontaining molecule comprising a first fibronectin type III (FN3) domainand a second FN3 domain, wherein the first FN3 domain specifically bindsepidermal growth factor receptor (EGFR) and blocks binding of epidermalgrowth factor (EGF) to EGFR, and the second FN3 domain specificallybinds hepatocyte growth factor receptor (c-Met), and blocks binding ofhepatocyte growth factor (HGF) to c-Met, wherein

the first FN3 domain comprises

-   -   an FG loop comprising the sequence HNVYKDTNX₉RGL (SEQ ID        NO: 179) or the sequence LGSYVFEHDVML (SEQ ID NO: 180), wherein        X₉ is M or I; and    -   a BC loop comprising the sequence X₁X₂X₃X₄X₅X₆X₇X₈(SEQ ID NO:        181), wherein        -   X₁ is A, T, G or D;        -   X₂ is A, D, Y or W;        -   X₃ is P, D or N;        -   X₄ is L or absent;        -   X₅ is D, H, R, G, Y or W;        -   X₆ is G, D or A;        -   X₇ is A, F, G, H or D; and        -   X₈ is Y, F or L; and

the second FN3 domain comprises

-   -   a C strand and a CD loop comprising the sequence DSFX₁₀IRYX₁₁E        X₁₂X₁₃X₁₄X₁₅GX₁₆ (SEQ ID NO: 184), wherein        -   X₁₀ is W, F or V;        -   X₁₁ is D, F or L;        -   X₁₂ is V, F or L;        -   X₁₃ is V, L or T;        -   X₁₄ is V, R, G, L, T or S;        -   X₁₅ is G, S, A, T or K; and        -   X₁₆ is E or D; and    -   a F strand and a FG loop comprising the sequence        TEYX₁₇VX₁₈IX₁₉X₂₀V KGGX₂₁X₂₂SX₂₃ (SEQ ID NO: 185), wherein        -   X₁₇ is Y, W, I, V, G or A;        -   X₁₈ is N, T, Q or G;        -   X₁₉ is L, M, N or I;        -   X₂₀ is G or S;        -   X₂₁ is S, L, G, Y, T, R, H or K;        -   X₂₂ is I, V or L; and        -   X₂₃ is V, T, H, I, P, Y or L.

In another embodiment, the bispecific molecule comprises the first FN3domain that binds EGFR comprising the sequence:

LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNX₉RGL PLSAEFTT (SEQ ID NO: 182), or thesequence

LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈ DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV LGSYVFEHDVMLPLSAEFTT (SEQ ID NO: 183),

wherein in the SEQ ID NOs: 182 and 183;

-   -   X₁ is A, T, G or D;    -   X₂ is A, D, Y or W;    -   X₃ is P, D or N;    -   X₄ is L or absent;    -   X₅ is D, H, R, G, Y or W;    -   X₆ is G, D or A;    -   X₇ is A, F, G, H or D;    -   X₈ is Y, F or L; and    -   X₉ is M or I.

In another embodiment, the bispecific molecule comprises the second FN3domain that binds c-Met comprising the sequence

LPAPKNLVVSRVTEDSARLSWTAPDAAF DSFX₁₀IRYX₁₁E X₁₂X₁₃X₁₄X₁₅GX₁₆AIVLTVPGSERSYDLTGLKPG TEYX₁₇VX₁₈IX₁₉X₂₀VKGGX₂₁X₂₂SX₂₃PLSAEFTT (SEQ IDNO: 186),

wherein

-   -   X₁₀ is W, F or V; and    -   X₁₁ is D, F or L;    -   X₁₂ is V, F or L;    -   X₁₃ is V, L or T;    -   X₁₄ is V, R, G, L, T or S;    -   X₁₅ is G, S, A, T or K;    -   X₁₆ is E or D;    -   X₁₇ is Y, W, I, V, G or A;    -   X₁₈ is N, T, Q or G;    -   X₁₉ is L, M, N or I;    -   X₂₀ is G or S;    -   X₂₁ is S, L, G, Y, T, R, H or K;    -   X₂₂ is I, V or L; and    -   X₂₃ is V, T, H, I, P, Y or L.

Exemplary bispecific EGFR/c-Met FN3 domain containing molecules comprisethe amino acid sequences shown in SEQ ID NOs: 50-72, 106, 118-121,138-165, 170-178 or 190-193.

The bispecific EGFR/c-Met molecules of the invention comprise certainstructural characteristics associated with their functionalcharacteristics, such as inhibition of EGFR autophosphorylation, such asthe FG loop of the first FN3 domain that binds EGFR comprising thesequence HNVYKDTNX₉RGL (SEQ ID NO: 179) or the sequence LGSYVFEHDVML(SEQ ID NO: 180), wherein X₉ is M or I.

In one embodiment, the bispecific EGFR/c-Met FN3 domain containingmolecules of the invention

-   -   inhibit EGF-induced EGFR phosphorylation at EGFR residues        Tyrosine 1173 with an IC₅₀ value of less than about 8×10⁻⁷ M        when measured in H292 cells using 50 ng/mL human EGF;    -   inhibit HGF-induced c-Met phosphorylation at c-Met residue        Tyrosine 1349 with and IC₅₀ value of less than about 8.4×10 M        when measured in NCI-H441 cells using 100 ng/mL human HGF;    -   inhibit HGF-induced NCI-H292 cell proliferation with an IC₅₀        value of less than about 9.5×10⁻⁶M wherein the cell        proliferation is induced with 10% FBS containing 7.5 ng HGF;    -   bind EGFR with a K_(D) of less than about 2.0×10⁻⁸ M; or    -   bind c-Met with a K_(D) of less than about 2.0×10⁻⁸ M; wherein        the K_(D) is measured using surface plasmon resonance as        described in Example 3 or Example 5.

In another embodiment, the bispecific EGFR/c-Met FN3 domain containingmolecules of the invention

-   -   inhibit EGF-induced EGFR phosphorylation at EGFR residues        Tyrosine 1173 with and IC₅₀ of between about 4.2×10⁻⁹ M and        8×10⁻⁷ M when measured in H292 cells using 50 ng/mL human EGF;    -   inhibit HGF-induced c-Met phosphorylation at c-Met residues        Tyrosine 1349 with and IC₅₀ value of between about 2.4×10⁻⁸ M to        about 8.4×10⁻⁷ M when measured in NCI-H441 cells using 100 ng/mL        human HGF;    -   inhibit HGF-induced NCI-H292 cell proliferation with an IC₅₀        value between about 2.3×10⁻⁸ M to about 9.5×10⁻⁶M wherein the        cell proliferation is induced with 10% FBS containing 7.5 ng        HGF;    -   bind EGFR with a K_(D) of between about 2×10⁻¹⁰ M to about        2.0×10⁻⁸ M; or    -   bind c-Met with a K_(D) of between about 1×10⁻⁹ M to about        2.0×10⁻⁸ M, wherein    -   the K_(D) is measured using surface plasmon resonance as        described in Example 3 or Example 5.

In one embodiment, bispecific EGFR/c-Met molecules comprise theEGFR-binding FN3 domain comprising the sequence

LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNX₉RGL PLSAEFTT (SEQ ID NO: 182), wherein

-   -   X₁ is D;    -   X₂ is D;    -   X₃ is P;    -   X₄ is absent;    -   X₅ is H or W;    -   X₆ is A;    -   X₇ is F    -   X₈ is Y; and    -   X₉ is M or I; and

the c-Met-binding FN3 domain comprising the sequence

LPAPKNLVVSRVTEDSARLSWTAPDAAF DSFX₁₀IRYX₁₁E X₁₂X₁₃X₁₄X₁₅GX₁₆AIVLTVPGSERSYDLTGLKPG TEYX₁₇VX₁₈IX₁₉X₂₀VKGGX₂₁X₂₂SX₂₃ PLSAEFTT (SEQ IDNO: 186),

wherein

-   -   X₁₀ is W;    -   X₁₁ is F;    -   X₁₂ is F;    -   X₁₃ is V or L;    -   X₁₄ is G or S;    -   X₁₅ is S or K;    -   X₁₆ is E or D;    -   X₁₇ is V;    -   X₁₈ is N;    -   X₁₉ is L or M;    -   X₂₀ is G or S;    -   X₂₁ is S or K;    -   X₂₂ is I; and    -   X₂₃ is P.

Exemplary bispecific EGFR/c-Met molecules are those having the sequenceshown in SEQ ID NOs: 57, 61, 62, 63, 64, 65, 66, 67, 68 or 190-193.

The bispecific molecules of the invention may further comprisesubstitutions at one or more residue positions in the first FN3 domainand/or the second FN3 domain corresponding to positions 11, 14, 17, 37,46, 73 and 86 in Tencon (SEQ ID NO: 1) as described above, and asubstitution at position 29. Exemplary substitutions are substitutionsE11N, E14P, L17A, E37P, N46V, G73Y, E86I and D29E (numbering accordingto SEQ ID NO: 1). Skilled in the art will appreciate that other aminoacids can be used for substitutions, such as amino acids within a familyof amino acids that are related in their side chains as described infra.The generated variants can be tested for their stability and binding toEGFR and/or c-Met using methods herein.

In one embodiment, the bispecific EGFR/c-Met FN3 domain containingmolecule comprises the first FN3 domain that binds specifically EGFR andthe second FN3 domain that binds specifically c-Met, wherein the firstFN3 domain comprises the sequence:

LPAPKNLVVSX₂₄VTX₂₅DSX₂₆RLSWDDPX₂₇AFYX₂₈SFLIQYQX₂₉SEKVGEAIX₃₀LTVPGSERSYDLTGLKPGTEYTVSIYX₃₁VHNVYKDTNX₃₂RGLPLSAX₃₃FTT (SEQ ID NO: 187),wherein

X₂₄ is E, N or R;

X₂₅ is E or P;

X₂₆ is L or A;

X₂₇ is H or W;

X₂₈ is E or D;

X₂₉ is E or P;

X₃₀ is N or V;

X₃₁ is G or Y;

X₃₂ is M or I; and

X₃₃ is E or I;

and the second FN3 domain comprises the sequence:

LPAPKNLVVSX₃₄VTX₃₅DSX₃₆RLSWTAPDAAFDSFWIRYFX₃₇FX₃₈X₃₉X₄₀GX₄₁AIX₄₂LTVPGSERSYDLTGLKPGTEYVVNIX₄₃X₄₄VKGGX₄₅ISPPLSAX₄₆FTT (SEQ ID NO: 188);wherein

X₃₄ is E, N or R;

X₃₅ is E or P;

X₃₆ is L or A;

X₃₇ is E or P;

X₃₈ is V or L;

X₃₉ is G or S;

X₄₀ is S or K;

X₄₁ is E or D;

X₄₂ is N or V;

X₄₃ is L or M;

X₄₄ is G or S;

X₄₅ is S or K; and

X₄₆ is E or I.

In other embodiments, the bispecific EGFR/c-Met FN3 domain containingmolecule comprises the first FN3 domain comprising an amino acidsequence at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequence of SEQ ID NO: 27, andthe second FN3 domain comprising an amino acid sequence at least 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequence of SEQ ID NO: 41.

The bispecific EGFR/c-Met FN3 domain containing molecules of theinvention may be tailored to a specific affinity towards EGFR and c-Metto maximize tumor accumulation.

Another embodiment of the invention is an isolated bispecific FN3 domaincontaining molecule comprising a first fibronectin type III (FN3) domainand a second FN3 domain, wherein the first FN3 domain specifically bindsepidermal growth factor receptor (EGFR) and blocks binding of epidermalgrowth factor (EGF) to EGFR, and the second FN3 domain specificallybinds hepatocyte growth factor receptor (c-Met), and blocks binding ofhepatocyte growth factor (HGF) to c-Met, wherein the first FN3 domainand the second FN3 domain is isolated from a library designed based onTencon sequence of SEQ ID NO: 1.

The bispecific EGFR/c-Met FN3 domain containing molecule of theinvention can be generated by covalently coupling the EGFR-binding FN3domain and the c-Met binding FN3 domain of the invention using wellknown methods. The FN3 domains may be linked via a linker, for example alinker containing poly-glycine, glycine and serine, or alanine andproline. Exemplary linker include (GS)₂, (SEQ ID NO: 78), (GGGS)₂ (SEQID NO: 224), (GGGGS)₅ (SEQ ID NO: 79), (AP)₂ (SEQ ID NO: 80), (AP)₅ (SEQID NO: 81), (AP)₁₀ (SEQ ID NO: 82), (AP)₂₀ (SEQ ID NO: 83), A(EAAAK)₅AAA(SEQ ID NO: 84). The use of naturally occurring as well as artificialpeptide linkers to connect polypeptides into novel linked fusionpolypeptides is well known in the literature (Hallewell et al., J BiolChem 264, 5260-5268, 1989; Alfthan et al., Protein Eng. 8, 725-731,1995; Robinson & Sauer, Biochemistry 35, 109-116, 1996; U.S. Pat. No.5,856,456). The bispecific EGFR/c-Met molecules of the invention may belinked together from a C-terminus of the first FN3 domain to theN-terminus of the second FN3 domain, or from the C-terminus of thesecond FN3 domain to the N-terminus of the first FN3 domain. AnyEGFR-binding FN3 domain may be covalently linked to a c-Met-binding FN3domain. Exemplary EGFR-binding FN3 domains are domains having the aminoacid sequence shown in SEQ ID NOs: 18-29, 107-110, 122-137 or 194-211,and exemplary c-Met binding FN3 domains are domains having the aminoacid sequence shown in SEQ ID NOs: 32-49, 111-114 or 212-223. TheEGFR-binding FN3 domains to be coupled to a bispecific molecule mayadditionally comprise an initiator methionine (Met) at their N-terminus.

Variants of the bispecific EGFR/c-Met FN3 domain containing moleculesare within the scope of the invention. For example, substitutions can bemade in the bispecific EGFR/c-Met FN3 domain containing molecule as longas the resulting variant retains similar selectivity and potency towardsEGFR and c-Met when compared to the parent molecule. Exemplarymodifications are for example conservative substitutions that willresult in variants with similar characteristics to those of the parentmolecules. Conservative substitutions are those that take place within afamily of amino acids that are related in their side chains. Geneticallyencoded amino acids can be divided into four families: (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3)nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); and (4) uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. Alternatively, the amino acid repertoire can be grouped as(1) acidic (aspartate, glutamate); (2) basic (lysine, argininehistidine), (3) aliphatic (glycine, alanine, valine, leucine,isoleucine, serine, threonine), with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine,tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry,2nd ed, WH Freeman and Co., 1981). Non-conservative substitutions can bemade to the bispecific EGFR/c-Met FN3 domain containing molecule thatinvolves substitutions of amino acid residues between different classesof amino acids to improve properties of the bispecific molecules.Whether a change in the amino acid sequence of a polypeptide or fragmentthereof results in a functional homolog can be readily determined byassessing the ability of the modified polypeptide or fragment to producea response in a fashion similar to the unmodified polypeptide orfragment using the assays described herein. Peptides, polypeptides orproteins in which more than one replacement has taken place can readilybe tested in the same manner.

The bispecific EGFR/c-Met FN3 domain containing molecules of theinvention may be generated as dimers or multimers, for example, as ameans to increase the valency and thus the avidity of target moleculebinding. The multimers may be generated by linking one or moreEGFR-binding FN3 domain and one or more c-Met-binding FN3 domain to formmolecules comprising at least three individual FN3 domains that are atleast bispecific for either EGFR or c-Met, for example by the inclusionof an amino acid linker using well known methods.

Another embodiment of the invention is a bispecific FN3 domaincontaining molecule comprising a first fibronectin type III (FN3) domainand a second FN3 domain, wherein the first FN3 domain specifically bindsepidermal growth factor receptor (EGFR) and blocks binding of epidermalgrowth factor (EGF) to EGFR, and the second FN3 domain specificallybinds hepatocyte growth factor receptor (c-Met), and blocks binding ofhepatocyte growth factor (HGF) to c-Met comprising the amino acidsequence shown in SEQ ID NOs: 50-72, 106, 118-121, 138-165, 170-179 or190-193.

Half-Life Extending Moieties

The bispecific EGFR/c-Met FN3 domain containing molecules or themonospecific EGFR or c-Met binding FN3 domains of the invention mayincorporate other subunits for example via covalent interaction. In oneaspect of the invention, the bispecific EGFR/c-Met FN3 domain containingmolecules of the invention further comprise a half-life extendingmoiety. Exemplary half-life extending moieties are albumin, albuminvariants, albumin-binding proteins and/or domains, transferrin andfragments and analogues thereof, and Fc regions. An exemplaryalbumin-binding domain is shown in SEQ ID NO: 117 and an exemplaryalbumin variant is shown in SEQ ID NO: 189.

All or a portion of an antibody constant region may be attached to themolecules of the invention to impart antibody-like properties,especially those properties associated with the Fc region, such as Fceffector functions such as C1q binding, complement dependentcytotoxicity (CDC), Fc receptor binding, antibody-dependentcell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cellsurface receptors (e.g., B cell receptor; BCR), and can be furthermodified by modifying residues in the Fc responsible for theseactivities (for review; see Strohl, Curr Opin Biotechnol. 20, 685-691,2009).

Additional moieties may be incorporated into the bispecific molecules ofthe invention such as polyethylene glycol (PEG) molecules, such asPEG5000 or PEG20,000, fatty acids and fatty acid esters of differentchain lengths, for example laurate, myristate, stearate, arachidate,behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid,octadecanedioic acid, docosanedioic acid, and the like, polylysine,octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides)for desired properties. These moieties may be direct fusions with theprotein scaffold coding sequences and may be generated by standardcloning and expression techniques. Alternatively, well known chemicalcoupling methods may be used to attach the moieties to recombinantlyproduced molecules of the invention.

A pegyl moiety may for example be added to the bispecific ormonospecific molecules of the invention by incorporating a cysteineresidue to the C-terminus of the molecule and attaching a pegyl group tothe cysteine using well known methods. Exemplary bispecific moleculeswith the C-terminal cysteine are those having the amino acid sequenceshown in SEQ IN NO: 170-178.

Monospecific and bispecific molecules of the invention incorporatingadditional moieties may be compared for functionality by several wellknown assays. For example, altered properties of monospecific and/orbispecific molecules due to incorporation of Fc domains and/or Fc domainvariants may be assayed in Fc receptor binding assays using solubleforms of the receptors, such as the FcγRI, FcγRII, FcγRIII or FcRnreceptors, or using well known cell-based assays measuring for exampleADCC or CDC, or evaluating pharmacokinetic properties of the moleculesof the invention in in vivo models.

Polynucleotides, Vectors, Host Cells

The invention provides for nucleic acids encoding the EGFR-binding orc-Met binding FN3 domains or the bispecific EGFR/c-Met FN3 domaincontaining molecules of the invention as isolated polynucleotides or asportions of expression vectors or as portions of linear DNA sequences,including linear DNA sequences used for in vitrotranscription/translation, vectors compatible with prokaryotic,eukaryotic or filamentous phage expression, secretion and/or display ofthe compositions or directed mutagens thereof. Certain exemplarypolynucleotides are disclosed herein, however, other polynucleotideswhich, given the degeneracy of the genetic code or codon preferences ina given expression system, encode the EGFR-binding or c-Met binding FN3domains or the bispecific EGFR/c-Met FN3 domain containing molecules ofthe invention are also within the scope of the invention.

One embodiment of the invention is an isolated polynucleotide encodingthe FN3 domain specifically binding EGFR having the amino acid sequenceof SEQ ID NOs: 18-29, 107-110, 122-137 or 194-211.

One embodiment of the invention is an isolated polynucleotide encodingthe FN3 domain specifically binding c-Met having the amino acid sequenceof the sequence shown in SEQ ID NOs: 32-49, 111-114 or 212-223.

One embodiment of the invention is an isolated polynucleotide encodingthe bispecific EGFR/-c-Met FN3 domain containing molecule having theamino acid sequence of SEQ ID NOs: 50-72, 106, 118-121, 138-165, 170-179or 190-193.

One embodiment of the invention is an isolated polynucleotide comprisingthe polynucleotide sequence of SEQ ID NOs: 97, 98, 103, 104, 115, 116 or166-169.

The polynucleotides of the invention may be produced by chemicalsynthesis such as solid phase polynucleotide synthesis on an automatedpolynucleotide synthesizer and assembled into complete single or doublestranded molecules. Alternatively, the polynucleotides of the inventionmay be produced by other techniques such a PCR followed by routinecloning. Techniques for producing or obtaining polynucleotides of agiven known sequence are well known in the art.

The polynucleotides of the invention may comprise at least onenon-coding sequence, such as a promoter or enhancer sequence, intron,polyadenylation signal, a cis sequence facilitating RepA binding, andthe like. The polynucleotide sequences may also comprise additionalsequences encoding additional amino acids that encode for example amarker or a tag sequence such as a histidine tag or an HA tag tofacilitate purification or detection of the protein, a signal sequence,a fusion protein partner such as RepA, Fc or bacteriophage coat proteinsuch as pIX or pIII.

Another embodiment of the invention is a vector comprising at least onepolynucleotide of the invention. Such vectors may be plasmid vectors,viral vectors, vectors for baculovirus expression, transposon basedvectors or any other vector suitable for introduction of thepolynucleotides of the invention into a given organism or geneticbackground by any means. Such vectors may be expression vectorscomprising nucleic acid sequence elements that can control, regulate,cause or permit expression of a polypeptide encoded by such a vector.Such elements may comprise transcriptional enhancer binding sites, RNApolymerase initiation sites, ribosome binding sites, and other sitesthat facilitate the expression of encoded polypeptides in a givenexpression system. Such expression systems may be cell-based, orcell-free systems well known in the art.

Another embodiment of the invention is a host cell comprising the vectorof the invention. A monospecific EGFR-binding or c-Met binding FN3domain or the bispecific EGFR/c-Met FN3 domain containing molecule ofthe invention can be optionally produced by a cell line, a mixed cellline, an immortalized cell or clonal population of immortalized cells,as well known in the art. See, e.g., Ausubel, et al., ed., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.(1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual,2^(nd) Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane,Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989);Colligan, et al., eds., Current Protocols in Immunology, John Wiley &Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols inProtein Science, John Wiley & Sons, NY, N.Y., (1997-2001).

The host cell chosen for expression may be of mammalian origin or may beselected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, He G2, SP2/0,HeLa, myeloma, lymphoma, yeast, insect or plant cells, or anyderivative, immortalized or transformed cell thereof. Alternatively, thehost cell may be selected from a species or organism incapable ofglycosylating polypeptides, e.g. a prokaryotic cell or organism, such asBL21, BL21(DE3), BL21-GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174(DE3),and any of the natural or engineered E. coli spp, Klebsiella spp., orPseudomonas spp strains.

Another embodiment of the invention is a method of producing theisolated FN3 domain specifically binding EGFR or c-Met of the inventionor the isolated bispecific EGFR/c-Met FN3 domain containing molecule ofthe invention, comprising culturing the isolated host cell of theinvention under conditions such that the isolated FN3 domainspecifically binding EGFR or c-Met or the isolated bispecific EGFR/c-MetFN3 domain containing molecule is expressed, and purifying the domain ormolecule.

The FN3 domain specifically binding EGFR or c-Met or the isolatedbispecific EGFR/c-Met FN3 domain containing molecule of the inventioncan be purified from recombinant cell cultures by well-known methods,for example by protein A purification, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography, or high performance liquid chromatography (HPLC).

Uses of Bispecific EGFR/c-Met FN3 Domain Containing Molecules andEGFR-Binding or c-Met Binding FN3 Domains of the Invention

The bispecific EGFR/c-Met FN3 domain containing molecules, the EGFRbinding FN3 domains or the c-Met binding FN3 domains of the inventionmay be used to diagnose, monitor, modulate, treat, alleviate, helpprevent the incidence of, or reduce the symptoms of human disease orspecific pathologies in cells, tissues, organs, fluid, or, generally, ahost. The methods of the invention may be used to treat an animalpatient belonging to any classification. Examples of such animalsinclude mammals such as humans, rodents, dogs, cats and farm animals.

One aspect of the invention is a method for inhibiting growth orproliferation of cells that express EGFR and/or c-Met, comprisingcontacting the cells with the isolated bispecific EGFR/c-Met FN3 domaincontaining molecule, the EGFR binding FN3 domain or the c-Met bindingFN3 domain of the invention.

Another aspect of the invention is a method for inhibiting growth ormetastasis of EGFR and/or c-Met-expressing tumor or cancer cells in asubject comprising administering to the subject an effective amount ofthe isolated bispecific EGFR/c-Met FN3 domain containing molecule, theEGFR binding FN3 domain or the c-Met binding FN3 domain of the inventionso that the growth or metastasis of EGFR- and/or c-Met-expressing tumoror cancer cell is inhibited.

Another aspect of the invention is a method of treating a subject havingcancer, comprising administering a therapeutically effective amount ofthe isolated bispecific EGFR/c-Met FN3 domain containing molecule, theEGFR binding FN3 domain, or the c-Met binding FN3 domain of theinvention to a patient in need thereof for a time sufficient to treatthe cancer.

The bispecific EGFR/c-Met FN3 domain containing molecule, the EGFRbinding FN3 domain or the c-Met binding FN3 domain of the invention maybe used for treatment of any disease or disorder characterized byabnormal activation or production of EGFR, c-Met, EGF or other EGFRligand or HGF, or disorder related to EGFR or c-Met expression, whichmay or may not involve malignancy or cancer, where abnormal activationand/or production of EGFR, c-Met, EGF or other EGFR ligand, or HGF isoccurring in cells or tissues of a subject having, or predisposed to,the disease or disorder. The bispecific EGFR/c-Met FN3 domain containingmolecule, the EGFR binding FN3 domain or the c-Met binding FN3 domain ofthe invention may be used for treatment of tumors, including cancers andbenign tumors. Cancers that are amenable to treatment by the bispecificmolecules of the invention include those that overexpress EGFR and/orc-Met, cancers associated with elevated EGFR activity and/or expressionlevels (such as, for example, an EGFR activating mutation, an EGFR geneamplification, or ligand mediated EGFR activation) and elevated c-Metactivity and/or expression levels (such as, for example, a c-Metactivating mutation, a c-Met gene amplification, or HGF mediated c-Metactivation.

Exemplary EGFR activating mutations that may be associated with cancerinclude point mutations, deletion mutations, insertion mutations,inversions or gene amplifications that lead to an increase in at leastone biological activity of EGFR, such as elevated tyrosine kinaseactivity, formation of receptor homodimers and heterodimers, enhancedligand binding etc. Mutations can be located in any portion of an EGFRgene or regulatory region associated with an EGFR gene and includemutations in exon 18, 19, 20 or 21 or mutations in the kinase domain.Exemplary activating EGFR mutations are G719A, L861X (X being any aminoacid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, L858P or T790Msubstitutions, deletion of E746-A750, deletion of R748-P753, insertionof Ala between M766 and A767, insertion of SVA (Ser, Val, Ala) between5768 and V769, and insertion of NS (Asn, Ser) between P772 and H773.Other examples of EGFR activating mutations are known in the art (seee.g., U.S. Pat. Publ. No. US2005/0272083). Information about EGFR andother ErbB receptors including receptor homo- and hetero-dimers,receptor ligands, autophosphorylation sites, and signaling moleculesinvolved in ErbB mediated signaling is known in the art (see e.g., Hynesand Lane, Nature Reviews Cancer 5: 341-354, 2005).

Exemplary c-Met activating mutations include point mutations, deletionmutations, insertion mutations, inversions or gene amplifications thatlead to an increase in at least one biological activity of a c-Metprotein, such as elevated tyrosine kinase activity, formation ofreceptor homodimers and heterodimers, enhanced ligand binding etc.Mutations can be located in any portion of the c-Met gene or regulatoryregions associated with the gene, such as mutations in the kinase domainof c-Met. Exemplary c-Met activating mutations are mutations at residuepositions N375, V13, V923, R175, V136, L229, 5323, R988, S1058/T1010 andE168. Methods for detecting EGFR and c-Met mutations or geneamplifications are well known.

Exemplary cancers that are amenable to treatment by the bispecificEGFR/c-Met FN3 domain containing molecule, the EGFR binding FN3 domainor the c-Met binding FN3 domain of the invention include epithelial cellcancers, breast cancer, ovarian cancer, lung cancer, non-small cell lungcancer (NSCLC), lung adenocarcinoma, colorectal cancer, anal cancer,prostate cancer, kidney cancer, bladder cancer, head and neck cancer,ovarian cancer, pancreatic cancer, skin cancer, oral cancer, esophagealcancer, vaginal cancer, cervical cancer, cancer of the spleen,testicular cancer, gastric cancer, cancer of the thymus, colon cancer,thyroid cancer, liver cancer, or sporadic or hereditary papillary renalcarcinoma (PRCC).

The FN3 domains that specifically bind c-Met and block binding of HGF toc-Met of the invention may be for treatment of tumors, including cancersand benign tumors. Cancers that are amenable to treatment by the c-Metbinding FN3 domains of the invention include those that overexpressc-Met. Exemplary cancers that are amenable to treatment by the FN3domains of the invention include epithelial cell cancers, breast cancer,ovarian cancer, lung cancer, colorectal cancer, anal cancer, prostatecancer, kidney cancer, bladder cancer, head and neck cancer, ovariancancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer,vaginal cancer, cervical cancer, cancer of the spleen, testicularcancer, and cancer of the thymus.

The FN3 domains that specifically bind EGFR and blocks binding of EGF tothe EGFR of the invention may be used for treatment of tumors, includingcancers and benign tumors. Cancers that are amenable to treatment by theFN3 domains of the invention include those that overexpress EGFR orvariants. Exemplary cancers that are amenable to treatment by the FN3domains of the invention include epithelial cell cancers, breast cancer,ovarian cancer, lung cancer, colorectal cancer, anal cancer, prostatecancer, kidney cancer, bladder cancer, head and neck cancer, ovariancancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer,vaginal cancer, cervical cancer, cancer of the spleen, testicularcancer, and cancer of the thymus.

In some methods described herein, the bispecific EGFR/c-Met FN3 domaincontaining molecule, the EGFR binding FN3 domain or the c-Met bindingFN3 domain of the invention may be used to treat a subject with a cancerthat is resistant or has acquired resistance to treatment with one ormore EGFR inhibitors. Exemplary EGFR inhibitors for which cancer mayacquire resistance are anti-EGFR antibodies cetuximab (Erbitux®),pantinumumab (Vectibix®), matuzumab, nimotuzumab, small molecule EGFRinhibitors Tarceva® (erlotinib), IRESSA (gefitinib), EKB-569 (pelitinib,irreversible EGFR TKI), pan-ErbB and other receptor tyrosine kinaseinhibitors lapatinib (EGFR and HER2 inhibitor), pelitinib (EGFR and HER2inhibitor), vandetanib (ZD6474, ZACTIMA™, EGFR, VEGFR2 and RET TKI),PF00299804 (dacomitinib, irreversible pan-ErbB TKI), CI-1033(irreversible pan-erbB TKI), afatinib (BIBW2992, irreversible pan-ErbBTKI), AV-412 (dual EGFR and ErbB2 inhibitor) EXEL-7647 (EGFR, ErbB2,GEVGR and EphB4 inhibitor), CO-1686 (irreversible mutant-selective EGFRTKI), AZD9291 (irreversible mutant-selective EGFR TKI), and HKI-272(neratinib, irreversible EGFR/ErbB2 inhibitor). The methods describedherein may be used to treat cancer that is resistant or has acquiredresistance to treatment with gefitinib, erlotinib, afatinib, CO-1686,AZD9291 and/or cetuximab. Exemplary bispecific EGFR/c-Met FN3 domaincontaining molecules, the EGFR binding FN3 domains or the c-Met bindingFN3 domains that can be used are those described herein having aminoacid sequences shown in SEQ ID NOs: 18-29, 107-110, 122-137, 194-211,32-49, 111-114, 212-223, 50-72, 106, 118-121, 138-165, 170-178 or190-193.

Another aspect of the invention is a method of treating a subject havingcancer, comprising administering a therapeutically effective amount ofthe bispecific EGFR/c-Met FN3 domain containing molecule, the FN3 domainthat specifically bind c-Met or the FN3 domain that specifically bindEGFR to a patient in need thereof for a time sufficient to treat thecancer, wherein the subject is resistant or has acquired resistant totreatment with erlotinib, gefitinib, afatinib, CO-1686 (CAS number:1374640-70-6), AZD9291 or cetuximab.

Various qualitative and/or quantitative methods may be used to determineif a subject is resistant, has developed or is susceptible to developinga resistance to treatment with an EGFR inhibitor. Symptoms that may beassociated with resistance to an EGFR inhibitor include, for example, adecline or plateau of the well-being of the patient, an increase in thesize of a tumor, arrested or slowed decline in growth of a tumor, and/orthe spread of cancerous cells in the body from one location to otherorgans, tissues or cells. Re-establishment or worsening of varioussymptoms associated with cancer may also be an indication that a subjecthas developed or is susceptible to developing resistance to EGFRinhibitors, such as anorexia, cognitive dysfunction, depression,dyspnea, fatigue, hormonal disturbances, neutropenia, pain, peripheralneuropathy, and sexual dysfunction. The symptoms associated with cancermay vary according to the type of cancer. For example, symptomsassociated with cervical cancer may include abnormal bleeding, unusualheavy vaginal discharge, pelvic pain that is not related to the normalmenstrual cycle, bladder pain or pain during urination, and bleedingbetween regular menstrual periods, after sexual intercourse, douching,or pelvic exam. Symptoms associated with lung cancer may includepersistent cough, coughing up blood, shortness of breath, wheezing chestpain, loss of appetite, losing weight without trying and fatigue.

Symptoms for liver cancer may include loss of appetite and weight,abdominal pain, especially in the upper right part of abdomen that mayextend into the back and shoulder, nausea and vomiting, general weaknessand fatigue, an enlarged liver, abdominal swelling (ascites), and ayellow discoloration of the skin and the whites of eyes (jaundice). Oneskilled in oncology may readily identify symptoms associated with aparticular cancer type.

Others means to determine if a subject has developed a resistance to anEGFR inhibitor include examining EGFR phosphorylation, ERK1/2phosphorylation and/or AKT phosphorylation in cancer cells, whereincreased phosphorylation may be indicative that the subject hasdeveloped or is susceptible to developing resistance to an EGFRinhibitor. Methods of determining EGFR, ERK1/2 and/or AKTphosphorylation are well known and described herein. Identification of asubject who has developed a resistance to an EGFR inhibitor may involvedetection of elevated c-Met expression levels or elevated c-Metactivity, for example, arising from increased levels of circulating HGF,an activating mutation of the c-Met gene or a c-Met gene amplification.

Another embodiment of the invention is a method of treating NSCLC in apatient having an NSCLC tumor or tumor metastasis having an activatingEGFR mutation or EGFR gene amplification, comprising administering tothe patient a therapeutically effective amount of the bispecificEGFR/c-Met FN3 domain containing molecule, the EGFR binding FN3 domainor the c-Met binding FN3 domain of the invention.

The bispecific EGFR/c-Met FN3 domain containing molecule, the EGFRbinding FN3 domain or the c-Met binding FN3 domain of the invention canbe used to treat non-small cell lung cancer (NSCLC), which includessquamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Insome embodiments, cells of the NSCLC have an epithelial phenotype. Insome embodiments, the NSCLC has acquired resistance to treatment withone or more EGFR inhibitors.

In NSCLC, specific mutations in the EGFR gene are associated with highresponse rates (70-80%) to EGFR tyrosine kinase inhibitors (EGFR-TKIs).A 5 amino acid deletion in exon 19 or the point mutation L858R in EGFRare associated with EGFR-TKI sensitivity (Nakata and Gotoh, Expert OpinTher Targets 16:771-781, 2012). These mutations result in aligand-independent activation of the EGFR kinase activity. ActivatingEGFR mutations occur in 10-30% of NSCLC patients and are significantlymore common in East Asians, women, never smokers, and patients withadenocarcinoma histology (Janne and Johnson Clin Cancer Res 12(14Suppl): 4416s-4420s, 2006). EGFR gene amplification is also stronglycorrelated with response after EGFR-TKI treatment (Cappuzzo et al., JNatl Cancer Inst 97:643-55, 2005).

Although the majority of NSCLC patients with EGFR mutations initiallyrespond to EGFR TKI therapy, virtually all acquire resistance thatprevents a durable response. 50-60% of patients acquire resistance dueto a second-site point mutation in the kinase domain of EGFR (T790M).Nearly 60% of all tumors that become resistant to EGFR tyrosine kinaseinhibitors increase c-Met expression, amplify the c-Met gene, orincrease its only known ligand, HGF (Turke et al., Cancer Cell,17:77-88, 2010).

Another embodiments of the invention is a method of treating patienthaving cancer, comprising administering a therapeutically effectiveamount of the bispecific EGFR/c-Met FN3 domain containing molecule, theEGFR binding FN3 domain or the c-Met binding FN3 domain of the inventionto a patient in need thereof for a time sufficient to treat the cancer,wherein the cancer is associated with an EGFR activating mutation, anEGFR gene amplification, a c-Met activating mutation or a c-Met geneamplification.

In some embodiments the EGFR activating mutation is G719A, G719X (Xbeing any amino acid), L861X (X being any amino acid), L858R, E746K,L747S, E749Q, A750P, A755V, V765M, L858P or T790M substitution, deletionof E746-A750, deletion of R748-P753, insertion of Ala (A) between M766and A767, insertion of Ser, Val and Ala (SVA) between S768 and V769, andinsertion of Asn and Ser (NS) between P772 and H773.

Another embodiments of the invention is a method of treating patienthaving cancer, comprising administering a therapeutically effectiveamount of the bispecific EGFR/c-Met FN3 domain containing molecule, theEGFR binding FN3 domain or the c-Met binding FN3 domain of the inventionto a patient in need thereof for a time sufficient to treat the cancer,wherein the cancer is associated with an EGFR mutation L858R, T790M ordeletion of residues E746-A750 (del(E746, A750)), EGFR amplification orc-Met amplification.

In some embodiments, the cancer is associated with wild type EGFR andwild type c-Met.

In some embodiments, the cancer is associated with wild type EGFR andc-Met amplification.

In some embodiments, the cancer is associated with EGFR L858R and T790Mmutations and wild type c-Met.

In some embodiments, the cancer is associated with EGFR deletion del(E764, A750) and wild type c-Met.

In some embodiments, the cancer is associated with EGFR deletiondel(E764, A750) and c-Met amplification.

In some embodiments, the cancer is associated with EGFR deletiondel(E764, A750), EGFR amplification and c-Met amplification.

In some embodiments, the patient has a NSCLC associated with EGFR L858Rand T790M mutations and wild type c-Met.

In some embodiments, the patient has a NSCLC associated with EGFRamplification and wild type c-Met.

In some embodiments, the patient has a NSCLC associated with EGFRamplification and c-Met amplification.

In some embodiments, the patient has a NSCLC associated with EGFRdeletion del(E764, A750) and wild type c-Met.

In some embodiments, the patient has a NSCLC associated with EGFRdeletion del(E764, A750) and c-Met amplification. Amplification of EGFRor c-Met may be evaluated by standard methods, for example bydetermining the copy number of the EGFR or c-Met gene by southernblotting, FISH, or comparative genomic hybridization (CGH)

The terms “treat” or “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the development or spread of cancer. For purposes ofthis invention, beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve a desiredtherapeutic result. A therapeutically effective amount of the bispecificEGFR/c-Met FN3 domain containing molecule, the EGFR binding FN3 domainor the c-Met binding FN3 domain of the invention may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the bispecific EGFR/c-Met FN3 domaincontaining molecule, the EGFR binding FN3 domain or the c-Met bindingFN3 domain of the invention to elicit a desired response in theindividual. Exemplary indicators of an effective EGFR/c-Met therapeuticthat may decline or abate in association with resistance include, forexample, improved well-being of the patient, decrease or shrinkage ofthe size of a tumor, arrested or slowed growth of a tumor, and/orabsence of metastasis of cancer cells to other locations in the body.

Administration/Pharmaceutical Compositions

The invention provides for pharmaceutical compositions the bispecificEGFR/c-Met FN3 domain containing molecule, the EGFR binding FN3 domainor the c-Met binding FN3 domain of the invention and a pharmaceuticallyacceptable carrier. For therapeutic use, the bispecific EGFR/c-Met FN3domain containing molecules, the EGFR-binding FN3 domains or thec-Met-binding FN3 domains of the invention may be prepared aspharmaceutical compositions containing an effective amount of the domainor molecule as an active ingredient in a pharmaceutically acceptablecarrier. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the active compound is administered. Such vehiclescan be liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3%glycine can be used. These solutions are sterile and generally free ofparticulate matter. They may be sterilized by conventional, well-knownsterilization techniques (e.g., filtration). The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, stabilizing, thickening, lubricating and coloring agents, etc.The concentration of the molecules of the invention in suchpharmaceutical formulation can vary widely, i.e., from less than about0.5%, usually at least about 1% to as much as 15 or 20% by weight andwill be selected primarily based on required dose, fluid volumes,viscosities, etc., according to the particular mode of administrationselected. Suitable vehicles and formulations, inclusive of other humanproteins, e.g., human serum albumin, are described, for example, in e.g.Remington: The Science and Practice of Pharmacy, 21^(st) Edition, Troy,D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.

The mode of administration for therapeutic use of the bispecificEGFR/c-Met FN3 domain containing molecules, the EGFR binding FN3 domainsor the c-Met binding FN3 domains of the invention may be any suitableroute that delivers the agent to the host, such as parenteraladministration, e.g., intradermal, intramuscular, intraperitoneal,intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal,intravaginal, rectal), using a formulation in a tablet, capsule,solution, powder, gel, particle; and contained in a syringe, animplanted device, osmotic pump, cartridge, micropump; or other meansappreciated by the skilled artisan, as well known in the art. Sitespecific administration may be achieved by for example intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracerebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intracardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravascular, intravesical, intralesional, vaginal, rectal, buccal,sublingual, intranasal, or transdermal delivery.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 ml sterile buffered water, andbetween about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg ormore preferably, about 5 mg to about 25 mg, of the FN3 domain of theinvention.

The bispecific EGFR/c-Met FN3 domain containing molecules, the EGFRbinding FN3 domains or the c-Met binding FN3 domains of the inventionmay be administered to a patient by any suitable route, for exampleparentally by intravenous (IV) infusion or bolus injection,intramuscularly or subcutaneously or intraperitoneally. IV infusion canbe given over as little as 15 minutes, but more often for 30 minutes, 60minutes, 90 minutes or even 2 or 3 hours. The bispecific EGFR/c-Met FN3domain containing molecules, the EGFR binding FN3 domains or the c-Metbinding FN3 domains of the invention may also be injected directly intothe site of disease (e.g., the tumor itself). The dose given to apatient having a cancer is sufficient to alleviate or at least partiallyarrest the disease being treated (“therapeutically effective amount”)and may be sometimes 0.1 to 10 mg/kg body weight, for example 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, for example 15, 20,30, 40, 50, 60, 70, 80, 90 or 100 mg/kg. A fixed unit dose may also begiven, for example, 50, 100, 200, 500 or 1000 mg, or the dose may bebased on the patient's surface area, e.g., 400, 300, 250, 200, or 100mg/m². Usually between 1 and 8 doses, (e.g., 1, 2, 3, 4, 5, 6, 7 or 8)may be administered to treat cancer, but 10, 12, 20 or more doses may begiven. Administration of the bispecific EGFR/c-Met FN3 domain containingmolecules, the EGFR binding FN3 domains or the c-Met binding FN3 domainsof the invention may be repeated after one day, two days, three days,four days, five days, six days, one week, two weeks, three weeks, onemonth, five weeks, six weeks, seven weeks, two months, three months,four months, five months, six months or longer. Repeated courses oftreatment are also possible, as is chronic administration. The repeatedadministration may be at the same dose or at a different dose.

For example, a pharmaceutical composition of the bispecific EGFR/c-MetFN3 domain containing molecules, the EGFR binding FN3 domains or thec-Met binding FN3 domains of the invention for intravenous infusion maybe made up to contain about 200 ml of sterile Ringer's solution, andabout 8 mg to about 2400 mg, about 400 mg to about 1600 mg, or about 400mg to about 800 mg of the bispecific EGFR/c-Met antibody foradministration to a 80 kg patient. Methods for preparing parenterallyadministrable compositions are well known and are described in moredetail in, for example, “Remington's Pharmaceutical Science”, 15th ed.,Mack Publishing Company, Easton, Pa.

The bispecific EGFR/c-Met FN3 domain containing molecules, theEGFR-binding FN3 domains or the c-Met-binding FN3 domains of theinvention can be lyophilized for storage and reconstituted in a suitablecarrier prior to use. This technique has been shown to be effective withconventional protein preparations and art-known lyophilization andreconstitution techniques can be employed.

The bispecific EGFR/c-Met FN3 domain containing molecules, theEGFR-binding FN3 domains or the c-Met-binding FN3 domains of theinvention may be administered to a subject in a single dose or theadministration may be repeated, e.g. after one day, two days, threedays, five days, six days, one week, two weeks, three weeks, one month,five weeks, six weeks, seven weeks, two months or three months. Therepeated administration can be at the same dose or at a different dose.The administration can be repeated once, twice, three times, four times,five times, six times, seven times, eight times, nine times, ten times,or more.

The bispecific EGFR/c-Met FN3 domain containing molecules, theEGFR-binding FN3 domains or the c-Met-binding FN3 domains of theinvention may be administered in combination with a second therapeuticagent simultaneously, sequentially or separately. The second therapeuticagent may be a chemotherapeutic agent, an anti-angiogenic agent, or acytotoxic drug. When used for treating cancer, the bispecific EGFR/c-MetFN3 domain containing molecules, the EGFR-binding FN3 domains or thec-Met-binding FN3 domains may be used in combination with conventionalcancer therapies, such as surgery, radiotherapy, chemotherapy orcombinations thereof. Exemplary agents that can be used in combinationwith the FN3 domains of the invention are antagonists of HER2, HER3,HER4, VEGF, and protein tyrosine kinase inhibitors such as Iressa®(gefitinib) and Tarceva (erlotinib).

The bispecific EGFR/c-Met FN3 domain containing molecules, theEGFR-binding FN3 domains or the c-Met-binding FN3 domain may beadministered together with any one or more of the chemotherapeutic drugsor other anti-cancer therapeutics known to those of skill in the art.Chemotherapeutic agents are chemical compounds useful in the treatmentof cancer and include growth inhibitory agents or other cytotoxic agentsand include alkylating agents, anti-metabolites, anti-microtubuleinhibitors, topoisomerase inhibitors, receptor tyrosine kinaseinhibitors, angiogenesis inhibitors and the like. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andcyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-FU;folic acid analogues such as denopterin, methotrexate, pteropterin,trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine,thiamiprine, thioguanine; pyrimidine analogues such as ancitabine,azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,doxifluridine, enocitabine, floxuridine; androgens such as calusterone,dromostanolone propionate, epitiostanol, mepitiostane, testolactone;anti-adrenals such as aminoglutethimide, mitotane, trilostane; folicacid replenisher such as frolinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene;edatraxate; defofamine; demecolcine; diaziquone; elfornithine;elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine;pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; members of taxoid ortaxane family, such as paclitaxel (TAXOL® docetaxel (TAXOTERE®) andanalogues thereof; chlorambucil; gemcitabine; 6-thioguanine;mercaptopurine; methotrexate; platinum analogues such as cisplatin andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine;novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate;CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoic acid; esperamicins; capecitabine; inhibitors ofreceptor tyrosine kinases and/or angiogenesis, including sorafenib(NEXAVAR®), sunitinib (SUTENT®), pazopanib (VOTRIENT™), toceranib(PALLADIA™), vandetanib (ZACTIMA™), cediranib (RECENTIN®), regorafenib(BAY 73-4506), axitinib (AG013736), lestaurtinib (CEP-701), erlotinib(TARCEVA®), gefitinib (IRESSA™), BIBW 2992 (TOVOK™), lapatinib(TYKERB®), neratinib (HKI-272), and the like, and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. Alsoincluded in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogensincluding for example tamoxifen, raloxifene, aromatase inhibiting4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018,onapristone, and toremifene (FARESTON®); and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Other conventional cytotoxic chemical compounds as thosedisclosed in Wiemann et al., 1985, in Medical Oncology (Calabresi et aL,eds.), Chapter 10, McMillan Publishing, are also applicable to themethods of the present invention.

Exemplary agents that may be used in combination with the bispecificEGFR/c-Met FN3 domain containing molecules, the EGFR-binding FN3 domainsor the c-Met-binding FN3 domaininclude tyrosine kinase inhibitors andtargeted anti-cancer therapies such as Iressa® (gefitinib) and

Tarceva (erlotinib) and other antagonists of HER2, HER3, HER4 or VEGF.Exemplary HER2 antagonists include CP-724-714, HERCEPTIN™ (trastuzumab),OMNITARG™ (pertuzumab), TAK-165, lapatinib (EGFR and HER2 inhibitor),and GW-282974. Exemplary HER3 antagonists include anti-Her3 antibodies(see e.g., U.S. Pat. Publ. No. US2004/0197332). Exemplary HER4antagonists include anti-HER4 siRNAs (see e.g., Maatta et al., Mol BiolCell 17: 67-79, 2006. An exemplary VEGF antagonist is Bevacizumab(Avastin™).

When a small molecule is used in combination with the bispecificEGFR/c-Met FN3 domain containing molecules, the EGFR-binding FN3 domainsor the c-Met-binding FN3 domains of the invention, it is typicallyadministered more often, preferably once a day, but 2, 3, 4 or moretimes per day is also possible, as is every two days, weekly or at someother interval. Small molecule drugs are often taken orally butparenteral administration is also possible, e.g., by IV infusion orbolus injection or subcutaneously or intramuscularly. Doses of smallmolecule drugs may typically be from 10 to 1000 mg, or about 100, 150,200 or 250 mg.

When the bispecific EGFR/c-Met FN3 domain containing molecules, theEGFR-binding FN3 domains or the c-Met-binding FN3 domains of theinvention is administered in combination with a second therapeuticagent, the combination may take place over any convenient timeframe. Forexample, the bispecific EGFR/c-Met FN3 domain containing molecule, theEGFR-binding FN3 domain or the c-Met-binding FN3 domain of the inventionand the second therapeutic agent may be administered to a patient on thesame day, and even in the same intravenous infusion. However, thebispecific EGFR/c-Met FN3 domain containing molecule, the EGFR-bindingFN3 domain or the c-Met-binding FN3 domain of the invention and thesecond therapeutic agent may also be administered on alternating days oralternating weeks, fortnights or months, and so on. In some methods, thebispecific EGFR/c-Met FN3 domain containing molecule, the EGFR-bindingFN3 domain or the c-Met-binding FN3 domain of the invention and thesecond therapeutic agent are administered with sufficient proximity intime that they are simultaneously present (e.g., in the serum) atdetectable levels in the patient being treated. In some methods, anentire course of treatment of the bispecific EGFR/c-Met FN3 domaincontaining molecule, the EGFR-binding FN3 domain or the c-Met-bindingFN3 domain of the invention consisting of a number of doses over a timeperiod is followed or preceded by a course of treatment of the secondtherapeutic agent also consisting of a number of doses. In some methods,treatment with the bispecific EGFR/c-Met FN3 domain containing molecule,the EGFR-binding FN3 domain or the c-Met-binding FN3 domain of theinvention administered second is begun if the patient has resistance ordevelops resistance to the second therapeutic agent administeredinitially. The patient may receive only a single course or multiplecourses of treatment with one or both the bispecific EGFR/c-Met FN3domain containing molecule, the EGFR-binding FN3 domain or thec-Met-binding FN3 domain of the invention and the second therapeuticagent. A recovery period of 1, 2 or several days or weeks may be usedbetween administration of the bispecific EGFR/c-Met FN3 domaincontaining molecule, the EGFR-binding FN3 domain or the c-Met-bindingFN3 domain of the invention and the second therapeutic agent. When asuitable treatment regiment has already been established for the secondtherapeutic agent, that regimen may be used in combination with thebispecific EGFR/c-Met FN3 domain containing molecule, the EGFR-bindingFN3 domain or the c-Met-binding FN3 domain of the invention. Forexample, Tarceva® (erlotinib) is taken as a 100 mg or 150 mg pill once aday, and Iressa® (gefitinib) is taken as 250 mg tablet daily.

The bispecific EGFR/c-Met FN3 domain containing molecule, theEGFR-binding FN3 domain or the c-Met-binding FN3 domain of theinvention, optionally in combination with the second therapeutic agentmay be administered together with any form of radiation therapyincluding external beam radiation, intensity modulated radiation therapy(IMRT) and any form of radiosurgery including Gamma Knife, Cyberknife,Linac, and interstitial radiation (e.g. implanted radioactive seeds,GliaSite balloon), and/or with surgery. Combination with radiationtherapy can be especially appropriate for head and neck cancer and braintumors.

While having described the invention in general terms, the embodimentsof the invention will be further disclosed in the following examplesthat should not be construed as limiting the scope of the claims.

Example 1. Construction of Tencon Libraries

Tencon (SEQ ID NO: 1) is an immunoglobulin-like scaffold, fibronectintype III (FN3) domain, designed from a consensus sequence of fifteen FN3domains from human tenascin-C (Jacobs et al., Protein Engineering,Design, and Selection, 25:107-117, 2012; U.S. Pat. Publ. No.2010/0216708). The crystal structure of Tencon shows six surface-exposedloops that connect seven beta-strands. These loops, or selected residueswithin each loop, can be randomized in order to construct libraries offibronectin type III (FN3) domains that can be used to select novelmolecules that bind to specific targets.

Tencon:

(SEQ ID NO 1): LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTTConstruction of TCL1 Library

A library designed to randomize only the FG loop of Tencon (SEQ ID NO:1), TCL1, was constructed for use with the cis-display system (Jacobs etal., Protein Engineering, Design, and Selection, 25:107-117, 2012). Inthis system, a single-strand DNA incorporating sequences for a Tacpromoter, Tencon library coding sequence, RepA coding sequence,cis-element, and ori element is produced. Upon expression in an in vitrotranscription/translation system, a complex is produced of theTencon-RepA fusion protein bound in cis to the DNA from which it isencoded. Complexes that bind to a target molecule are then isolated andamplified by polymerase chain reaction (PCR), as described below.

Construction of the TCL1 library for use with cis-display was achievedby successive rounds of PCR to produce the final linear, double-strandedDNA molecules in two halves; the 5′ fragment contains the promoter andTencon sequences, while the 3′ fragment contains the repA gene and thecis- and ori elements. These two halves are combined by restrictiondigest in order to produce the entire construct. The TCL1 library wasdesigned to incorporate random amino acids only in the FG loop ofTencon, KGGHRSN (SEQ ID NO: 86). NNS codons were used in theconstruction of this library, resulting in the possible incorporation ofall 20 amino acids and one stop codon into the FG loop. The TCL1 librarycontains six separate sub-libraries, each having a different randomizedFG loop length, from 7 to 12 residues, in order to further increasediversity. Design of tencon-based libraries are shown in Table 2.

TABLE 2 Library BC Loop Design FG Loop Design WT Tencon TAPDAAFD*KGGHRSN** TCL1 TAPDAAFD* XXXXXXX XXXXXXXX XXXXXXXXX XXXXXXXXXXXXXXXXXXXXX XXXXXXXXXXXX TCL2 ######## #####S## *TAPDAAFD: residues22-28 of SEQ ID NO: 1; **KGGHRSN: SEQ ID NO: 86 Xrefers to degenerateamino acids encoded by NNS codons. #refers to the ″designed distributionof amino acids″ described in the text.

To construct the TCL1 library, successive rounds of PCR were performedto append the Tac promoter, build degeneracy into the FG loop, and addnecessary restriction sites for final assembly. First, a DNA sequencecontaining the promoter sequence and Tencon sequence 5′ of the FG loopwas generated by PCR in two steps. DNA corresponding to the full Tencongene sequence was used as a PCR template with primers POP2220 (SEQID NO:2) and TC5′toFG (SEQID NO: 3). The resulting PCR product from thisreaction was used as a template for the next round of PCR amplificationwith primers 130mer (SEQID NO: 4) and Tc5′toFG to complete the appendingof the 5′ and promoter sequences to Tencon. Next, diversity wasintroduced into the FG loop by amplifying the DNA product produced inthe first step with forward primer POP2222 (SEQID NO: 5), and reverseprimers TCF7 (SEQID NO: 6), TCF8 (SEQID NO: 7), TCF9 (SEQID NO: 8),TCF10 (SEQID NO: 9), TCF11 (SEQID N NO: 10), or TCF12 (SEQID NO: 11),which contain degenerate nucleotides. At least eight 100 μL PCRreactions were performed for each sub-library to minimize PCR cycles andmaximize the diversity of the library. At least 5 μg of this PCR productwere gel-purified and used in a subsequent PCR step, with primersPOP2222 (SEQ ID NO: 5) and POP2234 (SEQID NO: 12), resulting in theattachment of a 6×His tag and NotI restriction site to the 3′ end of theTencon sequence. This PCR reaction was carried out using only fifteenPCR cycles and at least 500 ng of template DNA. The resulting PCRproduct was gel-purified, digested with NotI restriction enzyme, andpurified by Qiagen column

The 3′ fragment of the library is a constant DNA sequence containingelements for display, including a PspOMI restriction site, the codingregion of the repA gene, and the cis- and ori elements. PCR reactionswere performed using a plasmid (pCR4Blunt) (Invitrogen) containing thisDNA fragment with M13 Forward and M13 Reverse primers. The resulting PCRproducts were digested by PspOMI overnight and gel-purified. To ligatethe 5′ portion of library DNA to the 3′ DNA containing the repA gene, 2pmol of 5′ DNA were ligated to an equal molar amount of 3′ repA DNA inthe presence of NotI and PspOMI enzymes and T4 ligase. After overnightligation at 37° C., a small portion of the ligated DNA was run on a gelto check ligation efficiency. The ligated library product was split intotwelve PCR amplifications and a 12-cycle PCR reaction was run withprimer pair POP2250 (SEQID NO: 13) and DidLigRev (SEQID NO: 14). The DNAyield for each sub-library of TCL1 library ranged from 32-34 μg.

To assess the quality of the library, a small portion of the workinglibrary was amplified with primers Tcon5new2 (SEQID NO: 15) and Tcon6(SEQID NO: 16), and was cloned into a modified pET vector vialigase-independent cloning. The plasmid DNA was transformed intoBL21-GOLD (DE3) competent cells (Stratagene) and 96 randomly pickedcolonies were sequenced using a T7 promoter primer. No duplicatesequences were found. Overall, approximately 70-85% of clones had acomplete promoter and Tencon coding sequence without frame-shiftmutation. The functional sequence rate, which excludes clones with STOPcodons, was between 59% and 80%.

Construction of TCL2 Library

TCL2 library was constructed in which both the BC and the FG loops ofTencon were randomized and the distribution of amino acids at eachposition was strictly controlled. Table 3 shows the amino aciddistribution at desired loop positions in the TCL2 library. The designedamino acid distribution had two aims. First, the library was biasedtoward residues that were predicted to be structurally important forTencon folding and stability based on analysis of the Tencon crystalstructure and/or from homology modeling. For example, position 29 wasfixed to be only a subset of hydrophobic amino acids, as this residuewas buried in the hydrophobic core of the Tencon fold. A second layer ofdesign included biasing the amino acid distribution toward that ofresidues preferentially found in the heavy chain HCDR3 of antibodies, toefficiently produce high-affinity binders (Birtalan et al., J Mol Biol377:1518-28, 2008; Olson et al., Protein Sci 16:476-84, 2007). Towardsthis goal, the “designed distribution” of Table 3 refers to thedistribution as follows: 6% alanine, 6% arginine, 3.9% asparagine, 7.5%aspartic acid, 2.5% glutamic acid, 1.5% glutamine, 15% glycine, 2.3%histidine, 2.5% isoleucine, 5% leucine, 1.5% lysine, 2.5% phenylalanine,4% proline, 10% serine, 4.5% threonine, 4% tryptophan, 17.3% tyrosine,and 4% valine. This distribution is devoid of methionine, cysteine, andSTOP codons.

TABLE 3 Residue Position* WT residues Distribution in the TCL2 library22 T designed distribution 23 A designed distribution 24 P 50% P +designed distribution 25 D designed distribution 26 A 20% A + 20% G +designed distribution 27 A designed distribution 28 F 20% F, 20% I, 20%L, 20% V, 20% Y 29 D 33% D, 33% E, 33% T 75 K designed distribution 76 Gdesigned distribution 77 G designed distribution 78 H designeddistribution 79 R designed distribution 80 S 100% S 81 N designeddistribution 82 P 50% P + designed distribution *residue numbering isbased on Tencon sequence of SEQ ID NO: 1

The 5′ fragment of the TCL2 library contained the promoter and thecoding region of Tencon (SEQ ID NO: 1), which was chemically synthesizedas a library pool (Sloning Biotechnology). This pool of DNA contained atleast 1×10¹¹ different members. At the end of the fragment, a BsaIrestriction site was included in the design for ligation to RepA.

The 3′ fragment of the library was a constant DNA sequence containingelements for display including a 6×His tag, the coding region of therepA gene, and the cis-element. The DNA was prepared by PCR reactionusing an existing DNA template (above), and primers LS1008 (SEQID NO:17) and DidLigRev (SEQID NO: 14). To assemble the complete TCL2 library,a total of 1 μg of BsaI-digested 5′ Tencon library DNA was ligated to3.5 μg of the 3′ fragment that was prepared by restriction digestionwith the same enzyme. After overnight ligation, the DNA was purified byQiagen column and the DNA was quantified by measuring absorbance at 260nm. The ligated library product was amplified by a 12-cycle PCR reactionwith primer pair POP2250 (SEQID NO: 13) and DidLigRev (SEQID NO: 14). Atotal of 72 reactions were performed, each containing 50 ng of ligatedDNA products as a template. The total yield of TCL2 working library DNAwas about 100 μg. A small portion of the working library was sub-clonedand sequenced, as described above for library TCL1. No duplicatesequences were found. About 80% of the sequences contained completepromoter and Tencon coding sequences with no frame-shift mutations.

Construction of TCL14 Library

The top (BC, DE, and FG) and the bottom (AB, CD, and EF) loops, e.g.,the reported binding surfaces in the FN3 domains are separated by thebeta-strands that form the center of the FN3 structure. Alternativesurfaces residing on the two “sides” of the FN3 domains having differentshapes than the surfaces formed by loops only are formed at one side ofthe FN3 domain by two anti-parallel beta-strands, the C and the Fbeta-strands, and the CD and FG loops, and is herein called theC-CD-F-FG surface.

A library randomizing an alternative surface of Tencon was generated byrandomizing select surface exposed residues of the C and F strands, aswell as portions of the CD and FG loops as shown in FIG. 1. A Tenconvariant, Tencon27 (SEQ ID NO: 99) having following substitutions whencompared to Tencon (SEQ ID NO: 1) was used to generate the library; E11RL17A, N46V, E86I. A full description of the methods used to constructthis library is described in US. Pat. Publ. No. US2013/0226834.

Example 2: Selection of Fibronectin Type III (FN3) Domains that BindEGFR and Inhibit EGF Binding

Library Screening

Cis-display was used to select EGFR binding domains from the TCL1 andTCL2 libraries. A recombinant human extracellular domain of EGFR fusedto an IgG1 Fc (R&D Systems) was biotinylated using standard methods andused for panning (residues 25-645 of full length EGFR of SEQ ID NO: 73).For in vitro transcription and translation (ITT), 2-6 μg of library DNAwere incubated with 0.1 mM complete amino acids, 1×S30 premixcomponents, and 30 μL of S30 extract (Promega) in a total volume of 100μL and incubated at 30° C. After 1 hour, 450 μL of blocking solution(PBS pH 7.4, supplemented with 2% bovine serum albumin, 100 μg/mLherring sperm DNA, and 1 mg/mL heparin) were added and the reaction wasincubated on ice for 15 minutes. EGFR-Fc:EGF complexes were assembled atmolar ratios of 1:1 and 10:1 EGFR to EGF by mixing recombinant human EGF(R&D Systems) with biotinylated recombinant EGFR-Fc in blocking solutionfor 1 hour at room temperature. For binding, 500 μL of blocked ITTreactions were mixed with 100 μL of EGFR-Fc:EGF complexes and incubatedfor 1 hour at room temperature, after which bound complexes were pulleddown with magnetic neutravidin or streptavidin beads (Seradyne). Unboundlibrary members were removed by successive washes with PBST and PBS.After washing, DNA was eluted from the bound complexes by heating to 65°C. for 10 minutes, amplified by PCR, and attached to a DNA fragmentencoding RepA by restriction digestion and ligation for further roundsof panning High affinity binders were isolated by successively loweringthe concentration of target EGFR-Fc during each round from 200 nM to 50nM and increasing the washing stringency. In rounds 4 and 5, unbound andweakly bound FN3 domains were removed by washing in the presence of a10-fold molar excess of non-biotinylated EGFR-Fc overnight in PBS.

Following panning, selected FN3 domains were amplified by PCR usingoligos Tcon5new2 (SEQID NO: 15) and Tcon6 (SEQID NO: 16), subcloned intoa pET vector modified to include a ligase independent cloning site, andtransformed into BL21-GOLD (DE3) (Stratagene) cells for solubleexpression in E. coli using standard molecular biology techniques. Agene sequence encoding a C-terminal poly-histidine tag was added to eachFN3 domain to enable purification and detection. Cultures were grown toan optical density of 0.6-0.8 in 2YT medium supplemented with 100 μg/mLcarbenicillin in 1-mL 96-well blocks at 37° C. before the addition ofIPTG to 1 mM, at which point the temperature was reduced to 30° C. Cellswere harvested approximately 16 hours later by centrifugation and frozenat −20° C. Cell lysis was achieved by incubating each pellet in 0.6 mLof BugBuster® HT lysis buffer (Novagen EMD Biosciences) with shaking atroom temperature for 45 minutes.

Selection of FN3 Domains that Bind EGFR on Cells

To assess the ability of different FN3 domains to bind EGFR in a morephysiological context, their ability to bind A431 cells was measured.A431 cells (American Type Culture Collection, cat. #CRL-1555)over-express EGFR with ˜2×10⁶ receptors per cell. Cells were plated at5,000/well in opaque black 96-well plates and allowed to attachovernight at 37° C., in a humidified 5% CO₂ atmosphere. FN3domain-expressing bacterial lysates were diluted 1,000-fold into FACSstain buffer (Becton Dickinson) and incubated for 1 hour at roomtemperature in triplicate plates. Lysates were removed and cells werewashed 3 times with 150 μL/well of FACS stain buffer. Cells wereincubated with 50 μL/well of anti-penta His-Alexa488 antibody conjugate(Qiagen) diluted 1:100 in FACS stain buffer for 20 minutes at roomtemperature. Cells were washed 3 times with 150 μL/well of FACS stainbuffer, after which wells were filled with 100 μL of FACS stain bufferand read for fluorescence at 488 nm using an Acumen eX3 reader.Bacterial lysates containing FN3 domains were screened for their abilityto bind A431 cells (1320 crude bacterial lysates for TCL1 and TCL2libraries) and 516 positive clones were identified, where binding was≥10-fold over the background signal. 300 lysates from the TCL14 librarywere screened for binding, resulting in 58 unique FN3 domain sequencesthat bind to EGFR.

SELECTION of FN3 Domains that Inhibit EGF Binding to EGFR on Cells

To better characterize the mechanism of EGFR binding, the ability ofvarious identified FN3 domain clones to bind EGFR in an EGF-competitivemanner was measured using A431 cells and run in parallel with the A431binding assay screen. A431 cells were plated at 5,000/well in opaqueblack 96-well plates and allowed to attach overnight at 37° C. in ahumidified 5% CO₂ atmosphere. Cells were incubated with 50 μL/well of1:1,000 diluted bacterial lysate for 1 hour at room temperature intriplicate plates. Biotinylated EGF (Invitrogen, cat. #E-3477) was addedto each well for a final concentration of 30 ng/mL and incubated for 10minutes at room temperature. Cells were washed 3 times with 150 μL/wellof FACS stain buffer. Cells were incubated with 50 μL/well ofstreptavidin-phycoerythrin conjugate (Invitrogen) diluted 1:100 in FACSstain buffer for 20 minutes at room temperature. Cells were washed 3times with 150 μL/well of FACS stain buffer, after which wells werefilled with 100 μL of FACS stain buffer and read for fluorescence at 600nm using an Acumen eX3 reader.

Bacterial lysates containing the FN3 domains were screened in the EGFcompetition assay described above. 1320 crude bacterial lysates fromTCL1 and TCL2 libraries were screened resulting in 451 positive clonesthat inhibited EGF binding by >50%.

Expression and Purification of Identified FN3 Domains Binding EGFR

His-tagged FN3 domains were purified from clarified E. coli lysates withHis MultiTrap™ HP plates (GE Healthcare) and eluted in buffer containing20 mM sodium phosphate, 500 mM sodium chloride, and 250 mM imidazole atpH 7.4. Purified samples were exchanged into PBS pH 7.4 for analysisusing PD MultiTrap™ G-25 plates (GE Healthcare).

Size Exclusion Chromatography Analysis

Size exclusion chromatography was used to determine the aggregationstate of the FN3 domains binding EGFR. Aliquots (10 μL) of each purifiedFN3 domain were injected onto a Superdex 75 5/150 column (GE Healthcare)at a flow rate of 0.3 mL/min in a mobile phase of PBS pH 7.4. Elutionfrom the column was monitored by absorbance at 280 nm. FN3 domains thatexhibited high levels of aggregation by SEC were excluded from furtheranalysis.

Off-Rate of Selected EGFR-Binding FN3 Domains from EGFR-Fc

Select EGFR-binding FN3 domains were screened to identify those withslow off-rates (k_(off)) in binding to EGFR-Fc on a ProteOn XPR-36instrument (Bio-Rad) to facilitate selection of high affinity binders.Goat anti-human Fc IgG (R&D systems), at a concentration of 5 μg/mL, wasdirectly immobilized via amine coupling (at pH 5.0) on all 6 ligandchannels in horizontal orientation on the chip with a flow rate of 30μL/min in PBS containing 0.005% Tween-20. The immobilization densitiesaveraged about 1500 Response Units (RU) with less than 5% variationamong different channels. EGFR-Fc was captured on the anti-human Fc IgGsurface to a density around 600 RU in vertical ligand orientation. Alltested FN3 domains were normalized to a concentration of 1 μM and testedfor their binding in horizontal orientation. All 6 analyte channels wereused for the FN3 domains to maximize screening throughput. Thedissociation phase was monitored for 10 minutes at a flow rate of 100μL/min. The inter-spot binding signals were used as references tomonitor non-specific binding between analytes and the immobilized IgGsurface, and were subtracted from all binding responses. The processedbinding data were locally fit to a 1:1 simple Langmuir binding model toextract the k_(off) for each FN3 domain binding to captured EGFR-Fc.

Inhibition of EGF-Stimulated EGFR Phosphorylation

Purified EGFR-binding FN3 domains were tested for their ability toinhibit EGF-stimulated phosphorylation of EGFR in A431 cells at a singleconcentration. EGFR phosphorylation was monitored using the EGFR phospho(Tyr1173) kit (Meso Scale Discovery). Cells were plated at 20,000/wellin clear 96-well tissue culture-treated plates (Nunc) in 100 μL/well ofRPMI medium (Gibco) containing GlutaMAX™ with 10% fetal bovine serum(FBS) (Gibco) and allowed to attach overnight at 37° C. in a humidified5% CO₂ atmosphere. Culture medium was removed completely and cells werestarved overnight in 100 μL/well of medium containing no FBS at 37° C.in a humidified 5% CO₂ atmosphere. Cells were then treated with 100μL/well of pre-warmed (37° C.) starvation medium containing EGFR-bindingFN3 domains at a concentration of 2 μM for 1 hour at 37° C. in ahumidified 5% CO₂ atmosphere. Controls were treated with starvationmedium only. Cells were stimulated by the addition and gentle mixing of100 μL/well of pre-warmed (37° C.) starvation medium containing 100ng/mL recombinant human EGF (R&D Systems, cat. #236-EG), for finalconcentrations of 50 ng/mL EGF and 1 μM EGFR-binding FN3 domain, andincubation at 37° C., 5% CO₂ for 15 minutes. One set of control wellswas left un-stimulated as negative controls. Medium was completelyremoved and cells were lysed with 100 μL/well of Complete Lysis Buffer(Meso Scale Discovery) for 10 minutes at room temperature with shaking,as per the manufacturer's instructions. Assay plates configured formeasuring EGFR phosphorylated on tyrosine 1173 (Meso Scale Discovery)were blocked with the provided blocking solution as per themanufacturer's instructions at room temperature for 1.5-2 hours. Plateswere then washed 4 times with 200 μL/well of 1× Tris Wash Buffer (MesoScale Discovery). Aliquots of cell lysate (30 μL/well) were transferredto assay plates, which were covered with plate sealing film (VWR) andincubated at room temperature with shaking for 1 hour. Assay plates werewashed 4 times with 200 μL/well of Tris Wash Buffer, after which 25 μLof ice-cold Detection Antibody Solution (Meso Scale Discovery) wereadded to each well, being careful not to introduce bubbles. Plates wereincubated at room temperature with shaking for 1 hour, followed by 4washes with 200 μL/well of Tris Wash Buffer. Signals were detected byaddition of 150 μL/well of Read Buffer (Meso Scale Discovery) andreading on a SECTOR® Imager 6000 instrument (Meso Scale Discovery) usingmanufacturer-installed assay-specific default settings. Percentinhibition of the EGF-stimulated positive control signal was calculatedfor each EGFR-binding FN3 domain.

Inhibition of EGF-stimulated EGFR phosphorylation was measured for 232identified clones from the TCL1 and TCL2 libraries. 22 of these clonesinhibited EGFR phosphorylation by ≥50% at 1 μM concentration. Afterremoval of clones that either expressed poorly or were judged to bemultimeric by size exclusion chromatography, nine clones were carriedforward for further biological characterization. The BC and FG loopsequences of these clones are shown in Table 4. Eight of the nineselected clones had a common FG loop sequence (HNVYKDTNMRGL; SEQ ID NO:95) and areas of significant similarity were seen between several clonesin their BC loop sequences.

TABLE 4  FN3 Domain BC Loop FG Loop SEQ SEQ SEQ ID ID ID Clone ID NO:Sequence NO: Sequence NO: P53A1R5-17 18 ADPHGFYD 87 HNVYKDTNMRGL 95P54AR4-17 19 TYDRDGYD 88 HNVYKDTNMRGL 95 P54AR4-47 20 WDPFSFYD 89HNVYKDTNMRGL 95 P54AR4-48 21 DDPRGFYE 90 HNVYKDTNMRGL 95 P54AR4-73 22TWPYADLD 91 HNVYKDTNMRGL 95 P54AR4-74 23 GYNGDHFD 92 HNVYKDTNMRGL 95P54AR4-81 24 DYDLGVYD 93 HNVYKDTNMRGL 95 P54AR4-83 25 DDPWDFYE 94HNVYKDTNMRGL 95 P54CR4-31 26 TAPDAAFD 85 LGSYVFEHDVM 96

Example 3: Characterization of EGFR-Binding FN3 Domains that Inhibit EGFBinding

Large-Scale Expression, Purification, and Endotoxin Removal

The FN3 domains shown in Table 4 were scaled up to provide more materialfor detailed characterization. An overnight culture containing eachEGFR-binding FN3 domain variant was used to inoculate 0.8 L of Terrificbroth medium supplemented with 100 μg/mL ampicillin at a 1/80 dilutionof overnight culture into fresh medium, and incubated with shaking at37° C. The culture was induced when the optical density at 600 nmreached ˜1.2-1.5 by addition of IPTG to a final concentration of 1 mMand the temperature was reduced to 30° C. After 4 hours, cells werecollected by centrifugation and the cell pellet was stored at −80° C.until needed.

For cell lysis, the thawed pellet was resuspended in 1× BugBuster®supplemented with 25 U/mL Benzonase® (Sigma-Aldrich) and 1 kU/mLrLysozyme™ (Novagen EMD Biosciences) at a ratio of 5 mL of BugBuster®per gram of pellet. Lysis proceeded for 1 hour at room temperature withgentle agitation, followed by centrifugation at 56,000×g for 50 minutesat 4° C. The supernatant was collected and filtered through a 0.2 μmfilter, then loaded on to a 5-mL HisTrap FF column pre-equilibrated withBuffer A (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 10 mM imidazole) using aGE Healthcare ÄKTAexplorer 100s chromatography system. The column waswashed with 20 column volumes of Buffer A and further washed with 16%Buffer B (50 mM Tris-HCl pH7.5, 500 mM NaCl, 250 mM imidazole) for 6column volumes. The FN3 domains were eluted with 50% B for 10 columnvolumes, followed by a gradient from 50-100% B over 6 column volumes.Fractions containing the FN3 domain protein were pooled, concentratedusing a Millipore 10K MWCO concentrator, and filtered before loadingonto a HiLoad™ 16/60 Superdex™ 75 column (GE Healthcare)pre-equilibrated with PBS. The protein monomer peak eluting from thesize exclusion column was retained.

Endotoxins were removed using a batch approach with ActiClean Etox resin(Sterogene Bioseparations). Prior to endotoxin removal, the resin waspre-treated with 1 N NaOH for 2 hours at 37° C. (or overnight at 4° C.)and washed extensively with PBS until the pH had stabilized to −7 asmeasured with pH indicator paper. The purified protein was filteredthrough a 0.2 μm filter before adding to 1 mL of Etox resin at a ratioof 10 mL of protein to 1 mL of resin. The binding of endotoxin to resinwas allowed to proceed at room temperature for at least 2 hours withgentle rotation. The resin was removed by centrifugation at 500×g for 2minutes and the protein supernatant was retained. Endotoxin levels weremeasured using EndoSafe®-PTS™ cartridges and analyzed on anEndoSafe®-MCS reader (Charles River). If endotoxin levels were above 5EU/mg after the first Etox treatment, the above procedure was repeateduntil endotoxin levels were decreased to ≤5 EU/mg. In cases where theendotoxin level was above 5 EU/mg and stabilized after two consecutivetreatments with Etox, anion exchange or hydrophobic interactionchromatography conditions were established for the protein to remove theremaining endotoxins.

Affinity Determination of Selected EGFR-Binding FN3 Domains to EGFR-Fc(EGFR-Fc Affinity)

Binding affinity of selected EGFR-binding FN3 domains to recombinantEGFR extracellular domain was further characterized by surface Plasmonresonance methods using a Proteon Instrument (BioRad). The assay set-up(chip preparation, EGFR-Fc capture) was similar to that described abovefor off-rate analysis. Selected EGFR binding FN3 domains were tested at1 μM concentration in 3-fold dilution series in the horizontalorientation. A buffer sample was also injected to monitor the baselinestability. The dissociation phase for all concentrations of eachEGFR-binding FN3 domain was monitored at a flow rate of 100 μL/min for30 minutes (for those with k_(off)˜10⁻² s⁻¹ from off-rate screening), or1 hour (for those with k_(off)˜10⁻³ s⁻¹ or slower). Two sets ofreference data were subtracted from the response data: 1) the inter-spotsignals to correct for the non-specific interactions between theEGFR-binding FN3 domain and the immobilized IgG surface; 2) the bufferchannel signals to correct for baseline drifting due to the dissociationof captured EGFR-Fc surface over time. The processed binding data at allconcentrations for each FN3 domain were globally fit to a 1:1 simpleLangmuir binding model to extract estimates of the kinetic (k_(on),k_(off)) and affinity (K_(D)) constants. Table 5 shows the kineticconstants for each of the constructs, with the affinity varying from 200pM to 9.6 nM.

Binding of Selected EGFR-Binding FN3 Domains to EGFR on Cells (“A431Cell Binding Assay”)

A431 cells were plated at 5,000/well in opaque black 96-well plates andallowed to attach overnight at 37° C., in a humidified 5% CO₂atmosphere. Purified EGFR-binding FN3 domains (1.5 nM to 30 μM) wereadded to the cells (in 50 uL) for 1 hour at room temperature intriplicate plates. Supernatant was removed and cells were washed 3 timeswith 150 μL/well of FACS stain buffer. Cells were incubated with 50μL/well of anti-penta His-Alexa488 antibody conjugate (Qiagen) diluted1:100 in FACS stain buffer for 20 minutes at room temperature. Cellswere washed 3 times with 150 μL/well of FACS stain buffer, after whichwells were filled with 100 μL of FACS stain buffer and read forfluorescence at 488 nm using an Acumen eX3 reader. Data were plotted asraw fluorescence signal against the logarithm of the FN3 domain molarconcentration and fitted to a sigmoidal dose-response curve withvariable slope using GraphPad Prism 4 (GraphPad Software) to calculateEC₅₀ values. Table 5 reports the EC₅₀ for each of the constructs rangingfrom 2.2 nM to >20 μM.

Inhibition of EGF Binding to EGFR on Cells Using Selected EGFR-BindingFN3 Domains (A431 Cell EGF Competition Assay)

A431 cells were plated at 5,000/well in opaque black 96-well plates andallowed to attach overnight at 37° C., in a humidified 5% CO₂atmosphere. Purified EGFR-binding FN3 domains (1.5 nM to 30 μM) wereadded to the cells (50 μL/well) for 1 hour at room temperature intriplicate plates. Biotinylated EGF (Invitrogen, Cat #: E-3477) wasadded to each well to give a final concentration of 30 ng/mL andincubated for 10 minutes at room temperature. Cells were washed 3 timeswith 150 μL/well of FACS stain buffer. Cells were incubated with 50μL/well of streptavidin-phycoerythrin conjugate (Invitrogen) diluted1:100 in FACS stain buffer for 20 minutes at room temperature. Cellswere washed 3 times with 150 μL/well of FACS stain buffer, after whichwells were filled with 100 μL of FACS stain buffer and read forfluorescence at 600 nm using an Acumen eX3 reader. Data were plotted asthe raw fluorescence signal against the logarithm of FN3 domain molarconcentration and fitted to a sigmoidal dose-response curve withvariable slope using GraphPad Prism 4 (GraphPad Software) to calculateIC₅₀ values. Table 5 reports the IC₅₀ values ranging from 1.8 nM to 121nM.

Inhibition of EGF-Stimulated EGFR Phosphorylation (Phoshpo-EGRF Assay)

Select FN3 domains that significantly inhibited EGF-stimulated EGFRphosphorylation were assessed more completely by measuring IC₅₀ valuesfor inhibition. Inhibition of EGF-stimulated EGFR phosphorylation wasassessed at varying FN3 domain concentrations (0.5 nM to 10 μM) asdescribed above in “inhibition of EGF stimulated EGFR phosphorylation”.Data were plotted as electrochemiluminescence signal against thelogarithm of the FN3 domain molar concentration and IC₅₀ values weredetermined by fitting data to a sigmoidal dose response with variableslope using GraphPad Prism 4 (GraphPad Software). Table 5 shows the IC₅₀values which ranged from 18 nM to >2.5 μM.

Inhibition of Human Tumor Cell Growth (NCI-H292 Growth and NCI-H322Growth Assay)

Inhibition of EGFR-dependent cell growth was assessed by measuringviability of the EGFR over-expressing human tumor cell lines, NCI-H292and NCI-H322 (American Type Culture Collection, cat. #CRL-1848 &#CRL-5806, respectively), following

TABLE 5 A431 NCI- NCI- EGFR- Cell A431 Phospho- H292 H322 FN3 Fc BindingCell EGF EGFR Growth Growth Domain SEQ ID Affinity EC₅₀ Competition IC₅₀IC₅₀ IC₅₀ Clone ID NO: (nM) (nM) IC₅₀ (nM) (nM) (nM) (nM) P53A1R5-17 181.89 4.0 9.8 >2500 86 65 P54AR4-17 19 9.62 16 21 184 ND ND P54AR4-47 202.51 8.6 7.1 295 44 39 P54AR4-48 21 7.78 12 9.8 170 ND ND P54AR4-73 220.197 9.4 4.6 141 83 73 P54AR4-74 23 ND 77 ND ND ND ND P54AR4-81 24 ND84 121 ND ND ND P54AR4-83 25 0.255 2.2 1.8 18 5.9 9.2 P54CR4-31 260.383 >20000 55 179 1150 >3073exposure to EGFR-binding FN3 domains. Cells were plated at 500cells/well (NCI-H292) or 1,000 cells/well (NCI-H322) in opaque white96-well tissue culture-treated plates (Nunc) in 100 μL/well of RPMImedium (Gibco) containing GlutaMAX™ and 10 mM HEPES, supplemented with10% heat inactivated fetal bovine serum (Gibco) and 1%penicillin/streptomycin (Gibco), and allowed to attach overnight at 37°C. in a humidified 5% CO₂ atmosphere. Cells were treated by addition of5 μL/well of phosphate-buffered saline (PBS) containing a concentrationrange of EGFR-binding FN3 domains. Controls were treated with 5 μL/wellof PBS only or 25 mM ethylenediaminetetraacetic acid in PBS. Cells wereincubated at 37° C., 5% CO₂ for 120 hours. Viable cells were detected byaddition of 75 μL/well of CellTiter-Glo® reagent (Promega), followed bymixing on a plate shaker for 2 minutes, and incubation in the dark atroom temperature for a further 10 minutes. Plates were read on aSpectraMax M5 plate reader (Molecular Devices) set to luminescence mode,with a read time of 0.5 seconds/well against a blank of medium only.Data were plotted as a percentage of PBS-treated cell growth against thelogarithm of FN3 domain molar concentration. IC₅₀ values were determinedby fitting data to the equation for a sigmoidal dose response withvariable slope using GraphPad Prism 4 (GraphPad Software). Table 5 showsIC₅₀ values ranging from 5.9 nM to 1.15 μM and 9.2 nM to >3.1 μM, usingthe NCI-H292 and NCI-H322 cells respectively. Table 5 shows the summaryof biological properties of EGFR-binding FN3 domains for each assay.

Example 4: Engineering of EGFR-Binding FN3 Domains

A subset of the EGFR binding FN3 domains was engineered to increase theconformational stability of each molecule. The mutations L17A, N46V andE86I which have been shown to improve FN3 domain stability (described inUS Pat. Publ. No. US2011/0274623) were incorporated into clonesP54AR4-83, P54CR4-31, and P54AR4-37 by DNA synthesis. The new mutants,P54AR5-83v2, P54CR431-v2, and P54AR4-37v2 were expressed and purified asdescribed above. Differential scanning calorimetry in PBS was used toassess the stability of each mutant in order to compare it to that ofthe corresponding parent molecule. Table 6 shows that each variantmolecule was stabilized significantly, with an average increase in theT_(m) of 18.5° C.

TABLE 6 FN3 domain Clone SEQID NO: T_(m) (° C.) P54AR4-83 25 50.6P54AR4-83v2 27 69.8 P54CR4-31 26 60.9 P54CR4-31v2 28 78.9 P54AR4-37 2245.9 P54AR4-37v2 29 64.2

Example 5: Selection of Fibronectin Type III (FN3) Domains that Bindc-Met and Inhibit HGF Binding

Panning on Human c-Met

The TCL14 library was screened against biotinylated-human c-Metextracellular domain (bt-c-Met) to identify FN3 domains capable ofspecifically binding c-Met. For selections, 3 μg of TCL14 library was invitro transcribed and translated (IVTT) in E. Coli S30 Linear Extract(Promega, Madison, Wis.) and the expressed library blocked with CisBlock (2% BSA (Sigma-Aldrich, St. Louis, Mo.), 100 μg/ml Herring SpermDNA (Promega), 1 mg/mL heparin (Sigma-Aldrich)). For selections,bt-c-Met was added at concentrations of 400 nM (Round 1), 200 nM (Rounds2 and 3) and 100 nM (Rounds 4 and 5). Bound library members wererecovered using neutravidin magnetic beads (Thermo Fisher, Rockford,Ill.) (Rounds 1, 3, and 5) or streptavidin magnetic beads (Promega)(Rounds 2 and 4) and unbound library members were removed by washing thebeads 5-14 times with 500 uL PBS-T followed by 2 washes with 500 μL PBS.

Additional selection rounds were performed to identify FN3 domainsmolecules with improved affinities. Briefly, outputs from round 5 wereprepared as described above and subjected to additional iterative roundsof selection with the following changes: incubation with bt-c-Met wasdecreased from 1 hour to 15 minutes and bead capture was decreased from20 minutes to 15 minutes, bt-c-Met decreased to 25 nM (Rounds 6 and 7)or 2.5 nM (Rounds 8 and 9), and an additional 1 hour wash was performedin the presence of an excess of non-biotinylated c-Met. The goal ofthese changes was to simultaneously select for binders with apotentially faster on-rate and a slower off-rate yielding asubstantially lower K_(D).

Rounds 5, 7 and 9 outputs were PCR cloned into a modified pET15 vector(EMD Biosciences, Gibbstown, N.J.) containing a ligase independentcloning site (pET15-LIC) using TCON6 (SEQID No. 30) and TCON5 E86I short(SEQID No. 31) primers, and the proteins were expressed as C-terminalHis6-tagged proteins after transformations and IPTG induction (1 mMfinal, 30° C. for 16 hours) using standard protocols. The cells wereharvested by centrifugation and subsequently lysed with Bugbuster HT(EMD Biosciences) supplemented with 0.2 mg/mL Chicken Egg White Lysozyme(Sigma-Aldrich). The bacterial lysates were clarified by centrifugationand the supernatants were transferred to new 96 deep-well plates.

Screening for FN3 Domains that Inhibit HGF Binding to c-Met

FN3 domains present in E. coli lysates were screened for their abilityto inhibit HGF binding to purified c-Met extracellular domain in abiochemical format. Recombinant human c-Met Fc chimera (0.5 μg/mL inPBS, 100 μL/well) was coated on 96-well White Maxisorp Plates (Nunc) andincubated overnight at 4° C. The plates were washed two times with 300μl/well of Tris-buffered saline with 0.05% Tween 20 (TBS-T,Sigma-Aldrich) on a Biotek plate washer. Assay plates were blocked withStartingBlock T20 (200 μL/well, Thermo Fisher Scientific, Rockland, IL)for 1 hour at room temperature (RT) with shaking and again washed twicewith 300 μl of TBS-T. FN3 domain lysates were diluted in StartingBlockT20 (from 1:10 to 1:100,000) using the Hamilton STARplus roboticssystem. Lysates (50 μL/well) were incubated on assay plates for 1 hourat RT with shaking. Without washing the plates, bt-HGF (1 μg/mL inStartingBlock T20, 50 μL/well, biotinylated) was added to the plate for30 min at RT while shaking. Control wells containing Tencon27 lysatesreceived either Starting Block T20 or diluted bt-HGF. Plates were thenwashed four times with 300 μl/well of TBS-T and incubated with 100μl/well of Streptavidin-HRP (1:2000 in TBS-T, Jackson Immunoresearch,West Grove, Pa.) for 30-40 minutes at RT with shaking. Again the plateswere washed four times with TBS-T. To develop signal, PODChemiluminescence Substrate (50 μL/well, Roche Diagnostics,Indianapolis, Ind.), prepared according to manufacturer's instructions,was added to the plate and within approximately 3 minutes luminescencewas read on the Molecular Devices M5 using SoftMax Pro. Percentinhibition was determined using the following calculation:100−((RLU_(sample)−Mean RLU_(No bt-HGF control))/(MeanRLU_(bt-HGF control)−Mean RLU_(No bt-HGF control))*100). Percentinhibition values of 50% or greater were considered hits.

High-Throughput Expression and Purification of FN3 Domains

His-tagged FN3 domains were purified from clarified E. coli lysates withHis MultiTrap™ HP plates (GE Healthcare) and eluted in buffer containing20 mM sodium phosphate, 500 mM sodium chloride, and 250 mM imidazole atpH 7.4. Purified samples were exchanged into PBS pH 7.4 for analysisusing PD MultiTrap™ G-25 plates (GE Healthcare).

IC₅₀ Determination of Inhibition of HGF Binding to c-Met

Select FN3 domains were further characterized in the HGF competitionassay. Dose response curves for purified FN3 domains were generatedutilizing the assay described above (starting concentrations of 5 μM).Percent inhibition values were calculated. The data were plotted as %inhibition against the logarithm of FN3 domain molar concentrations andIC₅₀ values were determined by fitting data to a sigmoidal dose responsewith variable slope using GraphPad Prism 4.

35 unique sequences were identified from Round 5 to exhibit activity atdilutions of 1:10, with IC₅₀ values ranging from 0.5 to 1500 nM. Round 7yielded 39 unique sequences with activity at dilutions of 1:100 and IC₅₀values ranging from 0.16 to 2.9 nM. 66 unique sequences were identifiedfrom Round 9, where hits were defined as being active at dilutions of1:1000. IC₅₀ values as low as 0.2 nM were observed in Round 9 (Table 8).

Affinity Determination of Selected c-Met-Binding FN3 Domains to c-Met-Fc(c-Met-Fc Affinity)

Affinities were determined from select c-Met binding FN3 domainsaccording to the protocol described for determination of affinities toEGFR binding FN3 domains in Example 3 except that c-Met-Fc fusionprotein was used in the experiments.

Example 6: Characterization of FN3 Domains that Bind c-Met and InhibitHGF Binding

FN3 domains were expressed and purified as described above in Example 2.Size exclusion chromatography and kinetic analysis was done as describedabove in Examples 1 and 2, respectively. Table 7 shows the sequences ofthe C-strand, CD loop, F-strand, and FG loop, and a SEQ ID NO: for theentire amino acid sequence for each domain.

TABLE 7 Clone SEQ ID Name NO: C loop CD strand F loop FG strandP114AR5P74-A5 32 FDSFWIRYDE VVVGGE TEYYVNILGV KGGSISV P114AR5P75-E9 33FDSFEIRYDE FLRSGE TEYWVTILGV KGGLVST P114AR7P92-F3 34 FDSFWIRYFE FLGSGETEYIVNIMGV KGGSISH P114AR7P92-F6 35 FDSFWIRYFE FLGSGE TEYVVNILGV KGGGLSVP114AR7P92-G8 36 FDSFVIRYFE FLGSGE TEYVVQILGV KGGYISI P114AR7P92-H5 37FDSFWIRYLE FLLGGE TEYVVQIMGV KGGTVSP P114AR7P93-D11 38 FDSFWIRYFE FLGSGETEYVVGINGV KGGYISY P114AR7P93-G8 39 FDSFWIRYFE FLGSGE TEYGVTINGV KGGRVSTP114AR7P93-H9 40 FDSFWIRYFE FLGSGE TEYVVQIIGV KGGHISL P114AR7P94-A3 41FDSFWIRYFE FLGSGE TEYVVNIMGV KGGKISP P114AR7P94-E5 42 FDSFWIRYFE FLGSGETEYAVNIMGV KGGRVSV P114AR7P95-B9 43 FDSFWIRYFE FLGSGE TEYVVQILGV KGGSISVP114AR7P95-D3 44 FDSFWIRYFE FLGSGE TEYVVNIMGV KGGSISY P114AR7P95-D4 45FDSFWIRYFE FLGSGE TEYVVQILGV KGGYISI P114AR7P95-E3 46 FDSFWIRYFE FLGSGETEYVVQIMGV KGGTVSP P114AR7P95-F10 47 FDSFWIRYFE FTTAGE TEYVVNIMGVKGGSISP P114AR7P95-G7 48 FDSFWIRYFE LLSTGE TEYVVNIMGV KGGSISPP114AR7P95-H8  49 FDSFWIRYFE FVSKGE TEYVVNIMGV KGGSISP C loop residuescorrespond to residues 28-37 of indicated SEQ ID NO: CD strand residuescorrespond to residues 38-43 of indicated SEQ ID NO: F loop residuescorrespond to residues 65-74 of indicated SEQ ID NO: FG strand residuescorrespond to residues 75-81 of indicated SEQ ID NO:Binding of Selected c-Met-Binding FN3 Domains to c-Met on Cells (“H441Cell Binding Assay”)

NCI-H441 cells (Cat # HTB-174, American Type Culture Collection,Manassas, Va.) were plated at 20,000 cells per well in Poly-D-lysinecoated black clear bottom 96-well plates (BD Biosciences, San Jose,Calif.) and allowed to attach overnight at 37° C., 5% CO₂. Purified FN3domains (50 μL/well; 0 to 1000 nM) were added to the cells for 1 hour at4° C. in duplicate plates. Supernatant was removed and cells were washedthree times with FACS stain buffer (150 μL/well, BD Biosciences, cat#554657). Cells were incubated with biotinylated-anti HIS antibody(diluted 1:160 in FACS stain buffer, 50 μL/well, R&D Systems, cat #BAM050) for 30 minutes at 4° C. Cells were washed three times with FACSstain buffer (150 μL/well), after which wells were incubated with antimouse IgG1-Alexa 488 conjugated antibody (diluted 1:80 in FACS stainbuffer, 50 μL/well, Life Technologies, cat # A21121) for 30 minutes at4° C. Cells were washed three times with FACS stain buffer (150 μL/well)and left in FACS stain buffer (50 μL/well). Total fluorescence wasdetermined using an Acumen eX3 reader. Data were plotted as rawfluorescence signal against the logarithm of the FN3 domain molarconcentration and fitted to a sigmoidal dose-response curve withvariable slope using GraphPad Prism 4 (GraphPad Software) to calculateEC₅₀ values. FN3 domains were found to exhibit a range of bindingactivities, with EC₅₀ values between 1.4 nM and 22.0 nM, as shown inTable 8.

Inhibition of HGF-Stimulated c-Met Phosphorylation

Purified FN3 domains were tested for their ability to inhibitHGF-stimulated phosphorylation of c-Met in NCI-H441, using the c-Metphospho(Tyr1349) kit from Meso Scale Discovery (Gaithersburg, Md.).Cells were plated at 20,000/well in clear 96-well tissue culture-treatedplates in 100 μL/well of RPMI medium (containing Glutamax and HEPES,Life Technologies) with 10% fetal bovine serum (FBS; Life Technologies)and allowed to attach overnight at 37° C., 5% CO₂. Culture medium wasremoved completely and cells were starved overnight in serum-free RPMImedium (100 μL/well) at 37° C., 5% CO₂. Cells were then replenished withfresh serum-free RPMI medium (100 μL/well) containing FN3 domains at aconcentration of 20 μM and below for 1 hour at 37° C., 5% CO₂. Controlswere treated with medium only. Cells were stimulated with 100 ng/mLrecombinant human HGF (100 μL/well, R&D Systems cat #294-HGN) andincubated at 37° C., 5% CO₂ for 15 minutes. One set of control wells wasleft un-stimulated as negative controls. Medium was then completelyremoved and cells were lysed with Complete Lysis Buffer (50 μL/well,Meso Scale Discovery) for 10 minutes at RT with shaking, as permanufacturer's instructions. Assay plates configured for measuringphosphorylated c-Met were blocked with the provided blocking solution asper the manufacturer's instructions at room temperature for 1 hour.Plates were then washed three times with Tris Wash Buffer (200 μL/well,Meso Scale Discovery). Cell lysates (30 μL/well) were transferred toassay plates, and incubated at RT with shaking for 1 hour. Assay plateswere then washed four times with Tris Wash Buffer, after which ice-coldDetection Antibody Solution (25 μL/well, Meso Scale Discovery) was addedto each well for 1 hr at RT with shaking. Plates were again rinsed fourtimes with Tris Wash Buffer. Signals were detected by addition of 150Read Buffer (150 μL/well, Meso Scale Discovery) and reading on a SECTOR®Imager 6000 instrument (Meso Scale Discovery) usingmanufacturer-installed assay-specific default settings. Data wereplotted as electrochemiluminescence signal against the logarithm of FN3domain molar concentration and IC₅₀ values were determined by fittingdata to a sigmoidal dose response with variable slope using GraphPadPrism 4. FN3 domains were found to inhibit phosphorylated c-Met withIC50 values ranging from 4.6 nM to 1415 nM as shown in Table 8.

Inhibition of Human Tumor Cell Growth

Inhibition of c-Met-dependent cell growth was assessed by measuringviability of U87-MG cells (American Type Culture Collection, cat #HTB-14), following exposure to c-Met-binding FN3 domains Cells wereplated at 8000 cells per well in opaque white 96-well tissueculture-treated plates (Nunc) in 100 μL/well of RPMI medium,supplemented with 10% FBS and allowed to attach overnight at 37° C., 5%CO₂. Twenty-four hours after plating, medium was aspirated and cellswere replenished with serum-free RPMI medium.

TABLE 8 pMet Inhbibition of HGF H441 Cell inhibition in Proliferation ofClone Affinity competition binding H441 cells U87-MG cells Name SEQ IDNO: (Kd, nM) IC50 (nM) (EC50, nM) (IC50, nM) (IC50, nM) P114AR5P74-A5 3210.1 5.2 18.7 1078 464.4 P114AR5P75-E9 33 45.8 51.9 ND 1415 1193.9P114AR7P92-F3 34 0.4 0.2 1.5 8.3 2.7 P114AR7P92-F6 35 3.1 2.2 4.9 165.3350.5 P114AR7P92-G8 36 1.0 1.6 5.9 155.3 123.9 P114AR7P92-H5 37 11.6 ND22.0 766.4 672.3 P114AR7P93-D11 38 ND ND 2.3 16 14.4 P114AR7P93-G8 396.9 1 3.8 459.5 103.5 P114AR7P93-H9 40 3.3 2.9 12.9 288.2 269.9P114AR7P94-A3 41 0.4 0.2 1.4 5 9.3 P114AR7P94-E5 42 4.2 0.7 3.4 124.3195.6 P114AR7P95-B9 43 0.5 0.3 ND 9.8 17.4 P114AR7P95-D3 44 0.3 0.2 1.54.6 1.7 P114AR7P95-D4 45 0.4 ND 1.4 19.5 19.4 P114AR7P95-E3 46 1.5 ND3.2 204.6 209.2 P114AR7P95-F10 47 4.2 1.4 4.4 187.6 129.7 P114AR7P95-G748 20.0 ND 11.3 659.3 692 P114AR7P95-H8 49 3.7 ND 4.1 209.8 280.7

Twenty-four hours after serum starvation, cells were treated by additionof serum-free medium containing c-Met-binding FN3 domains (30 μL/well).Cells were incubated at 37° C., 5% CO₂ for 72 hours. Viable cells weredetected by addition of 100 μL/well of CellTiter-Glo® reagent (Promega),followed by mixing on a plate shaker for 10 minutes. Plates were read ona SpectraMax M5 plate reader (Molecular Devices) set to luminescencemode, with a read time of 0.5 seconds/well. Data were plotted as rawluminescence units (RLU) against the logarithm of FN3 domain molarconcentration. IC₅₀ values were determined by fitting data to anequation for a sigmoidal dose response with variable slope usingGraphPad Prism 4. Table 8 reports IC₅₀ values ranging from 1 nM to >1000nM.

Characteristics of the c-Met binding FN3 domains are summarized in Table8.

Thermal Stability of c-Met-Binding FN3 Domains

Differential scanning calorimetry in PBS was used to assess thestability of each FN3 domain. Results of the experiment are shown inTable 9.

TABLE 9 Thermal Clone Stability Name SEQ ID NO: (Tm, C.) P114AR5P74-A532 74.1 P114AR5P75-E9 33 ND P114AR7P92-F3 34 81.5 P114AR7P92-F6 35 76.8P114AR7P92-G8 36 90.9 P114AR7P92-H5 37 87 P114AR7P93-D11 38 NDP114AR7P93-G8 39 76.8 P114AR7P93-H9 40 88.2 P114AR7P94-A3 41 86.2P114AR7P94-E5 42 80 P114AR7P95-B9 43 86.3 P114AR7P95-D3 44 82P114AR7P95-D4 45 85.3 P114AR7P95-E3 46 94.2 P114AR7P95-F10 47 85.2P114AR7P95-G7 48 87.2 P114AR7P95-H8 49 83

Example 7. Generation and Characterization of Bispecific Anti-EGFR/c-MetMolecules

Generation of Bispecific EGFR/c-Met Molecules

Numerous combinations of the EGFR and c-Met-binding FN3 domainsdescribed in Examples 1-6 were joined into bispecific molecules capableof binding to both EGFR and c-Met. Additionally, EGFR-binding FN3domains having amino acid sequences shown in SEQ ID NOs: 107-110 andc-Met binding FN3 domains having amino acid sequences shown in SEQ IDNOs: 111-114 were made and joined into bispecific molecules. Syntheticgenes were created to encode for the amino acid sequences described inSEQID NOs: 50-72, 106, 118-121 or 190-193 (Table 10) such that thefollowing format was maintained: EGFR-binding FN3 domain followed by apeptide linker followed by a c-Met-binding FN3 domain. A poly-histidinetag was incorporated at the C-terminus to aid purification. In additionto those molecules described in Table 10, the linker between the two FN3domains was varied according to length, sequence composition andstructure according to those listed in Table 11. It is envisioned that anumber of other linkers could be used to link such FN3 domainsBispecific EGFR/c-Met molecules were expressed and purified from E. colias described for monospecific EGFR or c-Met FN3 domains using IMAC andgel filtration chromatography steps. It is evident to the skilled in artthat the bispecific EGFR/c-Met molecules may or may not contain aninitiator methionine. Exemplary molecules with the initiator methionineare molecules having the amino acid sequence shown in SEQ ID NOs: 106,118-121, 138-165, 190 and 192, and exemplary molecules without theinitiator methionine are shown in SEQ ID NOs: 50-72, 191 and 193. Thepresence of the initiator methionine for the EGFR binding FN3 domainsensures proper activity; the initiator methionine has less impact on thec-Met FN3 domains.

TABLE 10 Bispecific EGFR/ EGFR-binding cMET-binding c-Met moleculeFN3 comain FN3 domain Linker Clone SEQ ID Clone SEQ ID Clone SEQ IDSEQ ID ID NO: ID NO: ID NO: Sequence NO: ECB1  50 P54AR4-83v2  27P114AR5P74-A5  32 (GGGGS)₄ 79 ECB2  51 P54AR4-83v2  27 P114AR7P94-A3  41(GGGGS)₄ 79 ECB3  52 P54AR4-83v2  27 P114AR7P93-H9  40 (GGGGS)₄ 79 ECB4 53 P54AR4-83v2  27 P114AR5P75-E9  33 (GGGGS)₄ 79 ECB5  54 P53A1R5-17V2107 P114AR7P94-A3  41 (GGGGS)₄ 79 ECB6  55 P53A1R5-17V2 107P114AR7P93-H9  40 (GGGGS)₄ 79 ECB7  56 P53A1R5-17V2 107 P114AR5P75-E9 33 (GGGGS)₄ 79 ECB15  57 P54AR4-83v2  27 P114AR7P94-A3  41 (AP)₅ 81ECB27  58 P54AR4-83v2  27 P114AR5P74-A5  32 (AP)₅ 81 ECB60  59P53A1R5-17V2 107 P114AR7P94-A3  41 (AP)₅ 81 ECB37  60 P53A1R5-17V2 107P114AR5P74-A5  32 (AP)₅ 81 ECB94  61 P54AR4-83v22 108 P114AR7P94-A3V22111 (AP)₅ 81 ECB95  62 P54AR4-83v22 108 P114AR9P121-A6v2 112 (AP)₅ 81ECB96  63 P54AR4-83v22 108 P114AR9P122-A7v2 113 (AP)₅ 81 ECB97  64P54AR4-83v22 108 P114AR7P95-C5V2 114 (AP)₅ 81 ECB106  65 P54AR4-83v23109 P114AR7P94-A3V22 111 (AP)₅ 81 ECB107  66 P54AR4-83v23 109P114AR9P121-A6v2 112 (AP)₅ 81 ECB108  67 P54AR4-83v23 109P114AR9P122-A7v2 113 (AP)₅ 81 ECB109  68 P54AR4-83v23 109P114AR7P95-C5V2 114 (AP)₅ 81 ECB118  69 P53A1R5-17V22 110P114AR7P94-A3V22 111 (AP)₅ 81 ECB119  70 P53A1R5-17V22 110P114AR9P121-A6v2 112 (AP)₅ 81 ECB120  71 P53A1R5-17V22 110P114AR9P122-A7v2 113 (AP)₅ 81 ECB121  72 P53A1R5-17V22 110P114AR7P95-C5V2 114 (AP)₅ 81 ECB91 106 P54AR4-83v22 108 P114AR7P95-C5V2114 (AP)₅ 81 ECB18 118 P54AR4-83v2  27 P114AR5P74-A5  32 (AP)₅ 81 ECB28119 P53A1R5-17V2 107 P114AR5P74-A5  32 (AP)₅ 81 ECB38 120 P54AR4-83v2 27 P114AR7P94-A3  41 (AP)₅ 81 ECB39 121 P53A1R5-17V2 107 P114AR7P94-A3 41 (AP)₅ 81 ECB168 190 P54AR4-83v22 108 P114AR7P95-C5V2 114 (AP)₅ 81ECB176 192 P53A1R5-17V2 107 P114AR7P95-C5V2 114 (AP)₅ 81

TABLE 11 SEQ ID Linker ength in Linker NO: amino acids Structure GS  78 2 Disordered GGGGS 105  5 Disordered (GGGGS)₂ 224 10 Disordered(GGGGS)₄  79 20 Disordered (AP)₂  80  4 Rigid (AP)₅  81  5 Rigid (AP)₁₀ 82 20 Rigid (AP)₂₀  83 40 Rigid A(EAAAK)₅AAA  84 29 α-helicalBispecific EGFR/c-Met Molecules Enhance Potency Compared to MonospecificMolecules Alone, Suggesting Avidity

NCI-H292 cells were plated in 96 well plates in RPMI medium containing10% FBS. 24 hours later, medium was replaced with serum free RPMI. 24hours after serum starvation, cells were treated with varyingconcentrations of FN3 domains: either a high affinity monospecific EGFRFN3 domain (P54AR4-83v2), a weak affinity monospecific c-Met FN3 domain(P114AR5P74-A5), the mixture of the two monospecific EGFR and c-Met FN3domains, or a bispecific EGFR/c-Met molecules comprised of the lowaffinity c-Met FN3 domain linked to the high affinity EGFR FN3 domain(ECB1). Cells were treated for 1 h with the monospecific or bispecificmolecules and then stimulated with EGF, HGF, or a combination of EGF andHGF for 15 minutes at 37° C., 5% CO₂. Cells were lysed with MSD LysisBuffer and cell signaling was assessed using appropriate MSD Assayplates, according to manufacturer's instructions, as described above.

The low affinity c-Met FN3 domain inhibited phosphorylation of c-Metwith an IC₅₀ of 610 nM (FIG. 4). As expected the EGFR FN3 domain was notable to inhibit c-Met phosphorylation and the mixture of themono-specific molecules looked identical to the c-Met FN3 domain alone.However, the bi-specific EGFR/c-Met molecule inhibited phosphorylationof c-Met with an IC₅₀ of 1 nM (FIG. 4), providing more than a 2-logshift in improving potency relative to the c-Met monospecific alone.

The potential for the bispecific EGFR/c-Met molecule to enhance theinhibition of c-Met and/or EGFR phosphorylation through an avidityeffect was evaluated in multiple cell types with variable c-Met and EGFRdensities and ratios (FIG. 5). NCI-H292, NCI-H441, or NCI-H596 cellswere plated in 96 well plates in RPMI medium containing 10% FBS. 24hours later, medium was replaced with serum free RPMI. 24 hours afterserum starvation, cells were treated with varying concentrations ofeither monospecific EGFR-binding FN3 domain, monospecific c-Met FN3domain, or a bispecific EGFR/c-Met molecule (ECB5, comprised ofP53A1R5-17v2 and P114AR7P94-A3). Cells were treated for 1 h with themonospecific or bispecific molecules and then stimulated with EGF, HGF,or a combination of EGF and HGF for 15 minutes at 37° C., 5% CO₂. Cellswere lysed with MSD Lysis Buffer and cell signaling was assessed usingappropriate MSD Assay plates, according to manufacturer's instructions,as described above.

FIG. 5 (A-C) shows the inhibition of EGFR using a monospecificEGFR-binding FN3 domain compared to a bispecific EGFR/cMet molecule inthree different cell lines. To assess avidity in an EGFR phosphorylationassay, a medium affinity EGFR-binding FN3 domain (1.9 nM) (P53A1R5-17v2)was compared to a bispecific EGFR/c-Met molecule containing the sameEGFR-binding FN3 domain linked to a high-affinity c-Met-binding FN3domain (0.4 nM) (P114AR7P94-A3). In H292 and H596 cells, inhibition ofphosphorylation of EGFR was comparable for the monospecific andbispecific molecules (FIGS. 5A and 5B), likely because these cell lineshave a high ratio of EGFR to c-Met receptors. To test this theory,inhibition of EGFR phosphorylation was evaluated in NCI-H441 cells whichexhibit more c-Met receptors than EGFR. Treatment of NCI-H441 cells withthe bispecific EGFR/c-Met molecule decreased the IC₅₀ for inhibition ofEGFR phosphorylation compared to the monospecific EGFR-binding FN3domain by 30-fold (FIG. 5C).

The potential for enhanced potency with a bi-specific EGFR/c-Metmolecule was evaluated in a c-Met phosphorylation assay using a moleculewith a high affinity to EGFR (0.26 nM) and medium affinity to c-Met(10.1 nM). In both NCI-H292 and NCI-H596 cells, the inhibition ofphosphorylation of c-Met was enhanced with the bispecific moleculecompared to the monospecific c-Met-binding FN3 domain, by 134 and 1012fold, respectively (FIGS. 3D and 3E).

It was verified that the enhanced potency for inhibition of EGFR andc-Met phosphorylation with the bispecific EGFR/c-Met moleculestranslated into an enhanced inhibition of signaling and proliferation.For these experiments, the mixture of FN3 EGFR-binding and c-Met-bindingFN3 domains was compared to a bispecific EGFR/c-Met molecule. Asdescribed in Tables 12 and 13, the IC₅₀ values for ERK phosphorylation(Table 12) and proliferation of H292 cells (Table 13) were decreasedwhen cells were treated with the bispecific EGFR/c-Met molecule comparedto the mixture of the monospecific binders. The IC₅₀ for inhibition ofERK phosphorylation for the bi-specific EGFR/c-Met molecule was 143-foldlower relative to the mixture of the two monospecific EGFR and c-Met FN3domains, showing the effect of avidity to the potency of the moleculesin this assay. In Table 12, the monospecific EGFR- and c-Met binding FN3domains do not fully inhibit activity and therefore the IC₅₀ valuesshown should be considered lower limits. The proliferation assay wascompleted using different combinations EGFR and c-Met binding FN3domains either as a mixture or linked in a bispecific format. The IC₅₀for inhibition of proliferation for the bispecific EGFR/c-Met moleculewas 34-236-fold lower relative to the mixture of the monospecific parentEGFR or c-Met binding FN3 domains. This confirmed that the avidityeffect observed at the level of the receptors (FIG. 4 and FIG. 5)translates into an improvement in inhibiting cell signaling (Table 12)and cell proliferation (Table 13).

TABLE 12 Specificity of the FN3-domain IC50 (nM) (ERK molecule Clone #Type phosphorylation) EGFR P54AR4-83v2 monospecific >10,000 c-MetP114AR5P74-A5 monospecific 2366 EGFR or c-Met P54AR4-83v2 + mixture of798.4 P114AR5P74-A5 monospecific molecules EGFR and c-Met ECB1bispecific 5.6

TABLE 13 Fold increase IC50 for in IC50 for EGFR-binding mixture ofbispecific/ FN3 domain c-Met binding FN3 monospecifics IC50 for mixtureof (affinity) domain (affinity) (nM) bispecific (nM) monospecificsP54AR4-83v2 P114ARP94-A3 36.5 1.04 35 (0.26 nM) (0.4 nM) P54AR4-83v2P114AR7P93-H9 274.5 8.05 34 (0.26 nM) (3.3 nM) P54AR4-83v2 P114AR5P74-A51719 7.29 236 (0.26 nM) (10.1 nM)In Vivo Tumor Xenografts: PK/PD

In order to determine efficacy of the monospecific and bispecific FN3domain molecules in vivo, tumor cells were engineered to secrete humanHGF (murine HGF does not bind to human c-Met). Human HGF was stablyexpressed in NCI-H292 cells using lentiviral infection (Lentiviral DNAvector expressing human HGF (Accession #X16322) and lentiviral packagingkit from Genecopoeia). After infection, HGF-expressing cells wereselected with 4 μg/mL puromycin (Invitrogen). Human HGF protein wasdetected in the conditioned medium of pooled cells using assay platesfrom MesoScale Discovery.

SCID Beige mice were subcutaneously inoculated with NCI-H292 cellsexpressing human HGF (2.0×10⁶ cells in Cultrex (Trevigen) in a volume of200 μL) on the dorsal flank of each animal Tumor measurements were takentwice weekly until tumor volumes ranged between 150-250 mm³ Mice werethen given a single i.p. dose of bispecific EGFR/c-Met molecules (linkedto an albumin binding domain to increase half-life) or PBS vehicle. At 6h or 72 h after dosing, tumors were extracted and immediately frozen inliquid nitrogen. Blood samples were collected via cardiac puncture into3.8% citrate containing protease inhibitors Immediately aftercollection, the blood samples were centrifuged and the resulting plasmawas transferred to sample tubes and stored at −80° C. Tumors wereweighed, cut into small pieces, and lysed in Lysing Matrix A tubes (LMA)containing RIPA buffer with HALT protease/phosphatase inhibitors(Pierce), 50 mM sodium fluoride (Sigma), 2 mM activated sodiumorthovanadate (Sigma), and 1 mM PMSF (MesoScale Discovery). Lysates wereremoved from LMA matrix and centrifuged to remove insoluble protein. Thesoluble tumor protein was quantified with a BCA protein assay anddiluted to equivalent protein levels in tumor lysis buffer.Phosphorylated c-Met, EGFR and ERK were measured using assay plates fromMesoScale Discovery (according to Manufacturer's protocol and asdescribed above).

FIG. 6 shows the results of the experiments. Each bispecific EGFR/c-Metmolecule significantly reduced the levels of phosphorylated c-Met, EGFR,and ERK at both 6 h and 72 h. The data presented in FIG. 6 show theimportance of inhibiting both c-Met and EGFR simultaneously and how theaffinity of the bispecific EGFR/c-Met molecule for each receptor plays arole in inhibition of downstream ERK. The molecules containing the highaffinity EGFR-binding FN3 domains (P54AR4-83v2; shown as “8” in theFigure, K_(D)=0.26 nM) inhibited phosphorylation of EGFR to a largerextent compared to those containing the medium affinity EGFR-binding FN3domains (P53A1R5-17v2; shown as “17” in the figure K_(D)=1.9 nM) at both6 h and 72 h. All four bispecific molecules tested completely inhibitedphosphorylation of ERK at the 6 hour time point, regardless of affinity.At the 72 hour time point, the molecules containing the tight affinityc-Met-binding domain (P114AR7P94-A3; shown as “A3” in the figureK_(D)=0.4 nM) significantly inhibited phosphorylation of ERK compared tothe medium affinity c-Met-binding FN3 domain (P114AR5P74-A5; shown as“A5” in the Figure; K_(D)=10.1 nM; FIG. 6).

The concentration of each bispecific EGFR/c-Met molecule was measured at6 and 72 hours after dosing in the blood and in the tumor (FIG. 7).Interestingly, the bispecific molecule with the medium affinityEGFR-binding domain (P53A1R5-17v2; K_(D)=1.9 nM) but high affinityc-Met-binding FN3 domain (P114AR7P94-A3; K_(D)=0.4 nM) had significantlymore tumor accumulation at 6 hours relative to the other molecules,while the difference is diminished by 72 hours. It can be hypothesizedthat cells outside the tumor have lower levels of both EGFR and c-Metsurface expression and therefore the medium affinity EGFR moleculedoesn't bind to normal tissue as tightly compared to the higher affinityEGFR-binding FN3 domain. Therefore there is more free medium affinityEGFR-binding FN3 domain available to bind in the tumor. Therefore,identifying the appropriate affinities to each receptor may allow foridentification of a therapeutic with decreased systemic toxicities andincreased tumor accumulation.

Tumor Efficacy Studies with Bispecific EGFR/c-Met Molecules

SCID Beige mice were subcutaneously inoculated with NCI-H292 cellsexpressing human HGF (2.0×10⁶ cells in Cultrex (Trevigen) in 200 μL) inthe dorsal flank of each animal One week after implantation, mice werestratified into groups with equivalent tumor volumes (mean tumorvolume=77.9+/−1.7 mm³) Mice were dosed three times per week with thebispecific molecules and tumor volumes were recorded twice weekly. Tumorgrowth inhibition (TGI) was observed with four different bispecificmolecules, with variable affinities for c-Met and EGFR. FIG. 8 shows thebenefit of inhibiting both c-Met and EGFR as a delay in tumor growth wasobserved in the mice treated with molecules containing the high affinityEGFR-binding FN3 domain compared to the medium affinity EGFR-binding FN3domain when the c-Met-binding FN3 domain was medium affinity (open vs.closed triangles, P54AR4-83v2-P114AR5P74-A5 compared toP53A1R5-17-P114AR5P74-A5). In addition, the data shows the importance ofhaving a high affinity c-Met-binding FN3 domain as bispecific moleculescontaining either the high or medium affinity EGFR-binding FN3 domainbut high affinity c-Met-binding FN3 domain showed the most efficacy(dotted gray and black lines, P54AR4-83v2-P114AR7P94-A3 andP53A1R5-17v2-P114AR7P94-A3).

Efficacy of Bispecific Molecule and Other Inhibitors of EGFR and c-Met

The in vivo therapeutic efficacies of a bispecific EGFR/c-Met molecule(ECB38) and the small molecule inhibitors crizotinib (c-Met inhibitor)and erlotinib (EGFR inhibitor), cetuximab (anti-EGFR antibody), each asa single agent, and the combination of crizotnib and erlontinib, wereevaluated in the treatment of subcutaneous H292-HGF human lung cancerxenograft model in SCID/Beige mice.

The H292-HGF cells were maintained in vitro in RPMI1640 mediumsupplemented with fetal bovine serum (10% v/v), and L-glutamine (2 mM)at 37° C. in an atmosphere of 5% CO₂ in air. The cells were routinelysubcultured twice weekly by trypsin-EDTA treatment. The cells growing inan exponential growth phase were harvested and counted for tumorinoculation.

TABLE 14 Dose Dosing Planned Actual Group N Treatment (mg/kg) RouteSchedule Schedule 1 10 Vehicle  0 i.p. QD × 3 QD × 3 Control weeks weeks2 10 bispecific 25 i.p. 3 3 EGFR/ times/week × times/week × c-Met 3weeks 3 weeks molecule 3 10 Crizotinib 50 p.o. QD × 3 QD × 17 weeks days4 10 Erlotinib 50 p.o. QD × 2 QD × 3 weeks weeks 5 10 Crizotinib 50 p.o.QD × 3 QD × 3 weeks weeks 6 10 Cetuximab 1 mg/ i.p. Q4d*6 Q4d*6 mouse N:animal number; p.o.: oral administration; i.p.: intraperitonealinjection 3 times/week: doses were given on days 1, 3 and 5 of the week.QD: once daily Q4d: once every four days; the interval of thecombination of crizotinib and erlotinib was 0.5 hrs; dosing volume wasadjusted based on body weight (10 l/g); a: dosing was not given on day14 post grouping.

Each mouse was inoculated subcutaneously at the right flank region withH292-HGF tumor cells (2×10⁶) in 0.1 ml of PBS with cultrex (1:1) fortumor development. The treatments were started when the mean tumor sizereached 139 mm³. The test article administration and the animal numbersin each study group were shown in the following experimental designtable. The date of tumor cell inoculation was denoted as day 0. Table 14shows the treatment groups.

Before commencement of treatment, all animals were weighed and the tumorvolumes were measured. Since the tumor volume can affect theeffectiveness of any given treatment, mice were assigned into groupsusing randomized block design based upon their tumor volumes. Thisensures that all the groups are comparable at the baseline. Therandomized block design was used to assign experimental animals togroups. First, the experimental animals were divided into homogeneousblocks according to their initial tumor volume. Secondly, within eachblock, randomization of experimental animals to treatments wasconducted. Using randomized block design to assign experimental animalsensured that each animal had the same probability of being assigned to agiven treatment and therefore systematic error was reduced.

At the time of routine monitoring, the animals were checked for anyeffects of tumor growth and treatments on normal behavior, such asmobility, visual estimation of food and water consumption, body weightgain/loss (body weights were measured twice weekly), eye/hair mattingand any other abnormal effect.

The endpoint was whether tumor growth can be delayed or tumor bearingmice can be cured. Tumor size was measured twice weekly in twodimensions using a caliper, and the volume was expressed in mm³ usingthe formula: V=0.5 a×b² where a and b are the long and short diametersof the tumor, respectively. The tumor size was then used forcalculations of both T-C and T/C values. T-C was calculated with T asthe time (in days) required for the mean tumor size of the treatmentgroup to reach 1000 mm³, and C was the time (in days) for the mean tumorsize of the control group to reach the same size. The T/C value (inpercent) was an indication of antitumor efficacy; T and C were the meantumor volume of the treated and control groups, respectively, on a givenday. Complete tumor regression (CR) is defined as tumors that arereduced to below the limit of palpation (62.5 mm³). Partial tumorregression (PR) is defined as tumors that are reduced from initial tumorvolume. A minimum duration of CR or PR in 3 or more successive tumormeasurements is required for a CP or PR to be considered durable.

Animals for which the body weight loss exceeded 20%, or for which themean tumor size of the group exceeds 2000 mm³ were euthanized. The studywas terminated after two weeks of observation after the final dose.

Summary statistics, including mean and the standard error of the mean(SEM), are provided for the tumor volume of each group at each timepoint are shown in Table 15. Statistical analyses of difference in tumorvolume among the groups were evaluated using a one-way ANOVA followed byindividual comparisons using Games-Howell (equal variance not assumed).All data were analyzed using SPSS 18.0. p<0.05 was considered to bestatistically significant.

TABLE 15 Tumor volume (mm³)a bispecific Crizotinib; EGFR/c- ErlotinibMet Crizotinib at Cetuximab molecule at at Erlotinib at 50 mg/kg; at 1mg/ Days Vehicle 25 mg/kg 50 mg/kg 50 mg/kg 50 mg/kg mouse 7 139 ± 7 137 ± 7  140 ± 9  141 ± 8  139 ± 8  139 ± 10 9 230 ± 20 142 ± 7  217 ±20 201 ± 19 134 ± 9  168 ± 13 13 516 ± 45 83 ± 6 547 ± 43 392 ± 46 109 ±10 212 ± 20 16  808 ± 104 44 ± 7 914 ± 92 560 ± 70 127 ± 15 252 ± 28 201280 ± 209 30 ± 6 1438 ± 239  872 ± 136 214 ± 30 371 ± 48 23 1758 ± 25923 ± 7 2102 ± 298 1122 ± 202 265 ± 40 485 ± 61 27 2264 ± 318 21 ± 5 —1419 ± 577 266 ± 42 640 ± 82 30 — 23 ± 6 — 1516 ± 623 482 ± 61  869 ±100

The mean tumor size of the vehicle treated group (Group 1) reached 1,758mm³ at day 23 after tumor inoculation. Treatment with the bispecificEGFR/c-Met molecule at 25 mg/kg dose level (Group 2) led to completetumor regression (CR) in all mice which were durable in >3 successivetumor measurements (average TV=23 mm³, T/C value=1%, p=0.004 comparedwith the vehicle group at day 23).

Treatment with crizotinib as a single agent at 50 mg/kg dose level(Group 3) showed no antitumor activity; the mean tumor size was 2,102mm³ at day 23 (T/C value=120%, p=0.944 compared with the vehicle group).

Treatment with erlotinib as a single agent at 50 mg/kg dosing level(Group 4) showed minor antitumor activity, but no significant differencewas found compared with the vehicle group; the mean tumor size was 1,122mm³ at day 23 (T/C value=64%, p=0.429 compared with the vehicle group),with 4 days of tumor growth delay at tumor size of 1,000 mm³ comparedwith the vehicle group.

The combination of crizotinib (50 mg/kg, Group 5) and erlotinib (50mg/kg, Group 5) showed significant antitumor activity; the mean tumorsize was 265 mm³ at day 23 (T/C=15%; p=0.008), with 17 days of tumorgrowth delay at tumor size of 1,000 mm³ compared with the vehicle group.

Cetuximab at 1 mg/mouse dosing level as a single agent (Group 6) showedsignificant antitumor activities; the mean tumor size was 485 mm³ at day23 (T/C=28%; p=0.018), with 17 days of tumor growth delay at tumor sizeof 1,000 mm³ compared with the vehicle group. FIG. 9 and Table 16 showthe anti-tumor activities of the various therapies.

TABLE 16 Tumor Size T/C T-C (days) at P Treatment (mm³) at day 23 (%)1000 mm³ value Vehicle 1758 ± 259 — — — bispecific 23 ± 7 1 — 0.004EGFR/c-Met molecule (25 mg/kg) Crizotinib 2102 ± 298 120 −1 0.944 (50mg/kg) Erlotinib 1122 ± 202 64 4 0.429 (50 mg/kg) Crizotinib + 265 ± 4015 17 0.008 Erlotinib (50 mg/kg + 50 mg/kg) Cetuximab (1 mg/ 485 ± 61 2817 0.018 mouse)

Medium to severe body weight loss was observed in the vehicle groupwhich might be due to the increasing tumor burden; 3 mice died and 1mouse were euthanized when BWL>20% by day 23. Slight toxicity of thebispecific EGFR/c-Met molecule was observed in Group 2; 3 mice wereeuthanized when BWL>20% during the treatment period; the body weight wasgradually recovered when the treatment was withdrawn during the 2 weeksof observation period. More severe body weight loss was observed in thecrizotinib or erlotinib monotherapy group compared to the vehicle group,suggesting the treatment related toxicity. The combination of crizotiniband erlotinib was generally tolerated during the dosing phase, butsevere body weight loss was observed at the end of the study, whichmight be due to the resumption of the fast tumor growth during thenon-treatment period. The monotherapy of cetuximab was well tolerated inthe study; body weight loss was only observed at the end of the studydue to the resume of the tumor growth.

In summary, the bispecific EGFR/c-Met molecule at 25 mg/kg (3times/week×3 weeks) produced a complete response in H292-HGF human lungcancer xenograft model in SCID/Beige mice. The treatment was toleratedin 7 out of 10 mice, and resulted in severe body weight loss in 3 out of10 mice. FIG. 9 shows the impact of the various therapies on tumor sizeduring the time points after treatment.

Example 8: Half-Life Extension of the Bispecific EGFR/c-Met Molecules

Numerous methods have been described to reduce kidney filtration andthus extend the serum half-life of proteins including modification withpolyethylene glycol (PEG) or other polymers, binding to albumin, fusionto protein domains which bind to albumin or other serum proteins,genetic fusion to albumin, fusion to IgG Fc domains, and fusion to long,unstructured amino acid sequences.

Bispecific EGFR/c-Met molecules were modified with PEG in order toincrease the hydrodynamic radius by incorporating a free cysteine at theC-terminus of the molecule. Most commonly, the free thiol group of thecysteine residue is used to attach PEG molecules that are functionalizedwith maleimide or iodoacetemide groups using standard methods. Variousforms of PEG can be used to modify the protein including linear PEG of1000, 2000, 5000, 10,000, 20,000, or 40,000 kDa. Branched PEG moleculesof these molecular weights can also be used for modification. PEG groupsmay also be attached through primary amines in the bispecific EGFR/c-Metmolecules in some instances.

In addition to PEGylation, the half-life of bispecific EGFR/c-Metmolecules was extended by producing these proteins as fusion moleculeswith a naturally occurring 3-helix bundle serum albumin binding domain(ABD) or a consensus albumin binding domain (ABDCon). These proteindomains were linked to the C-terminus of the c-Met-binding FN3 domainvia any of the linkers described in Table 12. The ABD or ABDCon domainmay also be placed between the EGFR-binding FN3 domain and the c-Metbinding FN3 domain in the primary sequence. In some cases, albumin oralbumin variant (SEQ ID NO: 189) was linked to the bispecific EGFR/c-Metmolecules to the C-terminus of the c-Met binding FN3 domain.

Example 9: Characterization of Select Bispecific EGFR/c-Met Molecules

Select bispecific EGFR/c-Met molecules were characterized for theiraffinity to both EGFR and c-Met, their ability to inhibit EGFR and c-Metautophosphorylation, and their effect on proliferation of HGF cells.Binding affinity of the bispecific EGFR/c-Met molecules to recombinantEGFR and/or c-Met extracellular domain was further analyzed by surfacePlasmon resonance methods using a Proteon Instrument (BioRad) accordingto protocol described in Example 3. Results of the characterization areshown in Table 17.

TABLE 17 pMet pEGFR H292-HGF K_(D) inhibition inhibition ProliferationK_(D) (c- in in inhibition in HGF- (EGFR, Met, H441 cells H292 cellsinduced H292 cells Molecule nM) nM) (IC₅₀, nM) (IC₅₀, nM) (IC₅₀, nM)ECB15 0.2 2.6 n/a 4.2 23 ECB94 1 4.3 53.8 5.1 29.6 ECB95 1.1 6.2 178.813.6 383.4 ECB96 1.6 22.1 835.4 24.7 9480 ECB97 1.3 1.7 24.2 16.6 31.0ECB106 16.7 5.1 53.3 367.4 484.5 ECB107 16.9 9 29.9 812.3 2637 ECB10815.3 25.5 126.2 814.4 11372 ECB109 17.3 2.1 26 432 573.6 ECB168 0.4 0.323.1 ECB158* 0.9 0.58 10.8 *ECB158 is ECB168 conjugated to human serumalbumin variant C34S via a (GGGGS)₂ linker of SEQ ID NO: 224

Example 10. Paratopes of EGFR and c-Met Binding FN3 Domains

A series of mutations were made to molecule P54AR4-83v2 (SEQ Id NO: 27)in order to define residues critical for binding to the EGFRextracellular domain. For this analysis, every amino acid position inthe BC and FG loops were mutated to alanine one at a time to produce 18new molecules. The affinity that these mutants bind to EGFR wasdetermined by SPR analysis using a Proteon instrument. The results areshown in Table 18. 10 positions resulted in a loss of binding affinitygreater than 10-fold indicating that these positions contribute tobinding to EGFR. Fold change indicates fold change of the K_(D) value ofa variant when compared to the parent P54AR4-83v2. A combination ofresidues from the BC and FG loops makes up the binding surface. 10positions were shown to weaken binding to EGFR by greater than 10-fold,and 5 positions were shown to weaken binding to EGFR by greater than100-fold (D23, F27, Y28, V77, G85). In addition to P54AR4-83v2,EGFR-binding molecules P54AR4-48, P54AR4-81, P53A1R5-17v2, P54AR4-83v22and P54AR4-83v23 (SEQ ID NOs: 21, 25, 107, 108 and 109, respectively)have identical residues at paratope positions that weaken EGFR bindingby greater than 100-fold when mutated. Several bispecific EGFR/c-Metmolecules generated comprise the P54AR4-83v2, P54AR4-48, P54AR4-81,P53A1R5-17v2, P54AR4-83v22 or P54AR4-83v2 as their EGFR-binding FN3domain as shown in Table 10.

TABLE 18 SEQ ID Fold Molecule NO: k_(a) (1/Ms) k_(d) (1/s) K_(D) (nM)Change P54AR4-83v2 27 3.54E+05 4.98E−05 0.14 1 83v2 D22A 194 2.15E+053.01E−05 0.14 1 83v2 D23A 195 1.32E+05 4.20E−03 31.8 227 83v2 P24A 1967.81E+04 2.19E−04 2.8 20 83v2 W25A 197 1.10E+05 1.69E−04 1.5 11 83v2F27A 198 2.32E+04 5.56E−04 24 171 83v2 Y28A 199 4.36E+04 3.86E−03 88.5632 83v2 H75A 200 1.67E+05 6.55E−04 3.9 28 83v2 N76A 201 2.08E+057.43E−05 0.36 3 83v2 V77A 202 7.88E+04 8.55E−03 108 771 83v2 Y78A 2031.82E+05 5.14E−04 2.8 20 83v2 K79A 204 6.81E+05 2.83E−05 0.04 0 83v2D80A 205 1.23E+05 5.46E−05 0.45 3 83v2 M83A 206 1.77E+05 2.74E−04 1.5 1183v2 R84A 207 2.34E+05 1.37E−04 0.59 4 83v2 G85A 208 7.30E+04 2.20E−0330.1 215 83v2 L86A 209 3.09E+05 1.17E−04 0.38 3 83v2 T81A 210 2.28E+058.38E−05 0.37 3 83v2 N82A 211 1.94E+05 9.67E−05 0.5 4

Likewise, a series of mutations were made to the presumed c-Metinteraction surface of molecule P114AR7P95-A3 (SEQ ID NO: 41) in orderto define positions critical for target binding. This analysis was donein the context of bispecific molecule ECB15 (SEQ ID NO: 145) and serinewas used as a replacement instead of alanine as described above. Serinewas chosen to decrease the hydrophobicity of the resulting mutants.Table 19 describes the SPR results with the numbering of each mutationposition relative to that of molecule A3 (SEQ ID NO: 41). 7 positionswere shown to weaken binding to c-Met by greater than 10-fold. Nobinding was measurable for mutants M72S, R34S, and I79S. F38S mutationreduced binding to c-Met by greater than 100-fold. This datademonstrates that the positions contributing for c-Met binding aredistributed among the C-strand, F-strand, CD loop, and FG loop. Foldchange indicates fold change of the K_(D) value of a variant whencompared to the parent P114AR7P95-A3. In addition to P114AR7P94-A3,c-Met-binding molecules P114AR7P92-F3, P114AR7P95-D3, P114AR7P95-F10 andP114AR7P95-H8 (SEQ ID NOs: 34, 44, 47 and 49, respectively) haveidentical residues at paratope positions that weaken c-Met binding bygreater than 100-fold when mutated.

TABLE 19 SEQ KD Fold Sample ID NO: ka (1/Ms) kd (1/s) (nM) Change ECB15145 3.51E+05 1.33E−04 0.4 1 A3 K78S 212 4.40E+05 1.50E−04 0.3 0.75 A3G40S 213 1.85E+05 3.20E−04 1.7 4.25 A3 L39S 214 4.75E+05 1.27E−03 2.76.75 A3 V68S 215 3.29E+05 1.20E−03 3.6 9 A3 N70S 216 4.25E+05 2.49E−035.9 14.75 A3 P81S 217 3.21E+05 5.36E−04 1.7 4.25 A3 F36S 218 1.88E+055.12E−03 27.2 68 A3 W32S 219 2.89E+05 8.60E−03 29.8 74.5 A3 M72S 220 — —— A3 R34S 221 — — — A3 F38S 222 4.51E+04 3.23E—02 717 1792.5 A3 I79S 223— — —

Example 11. Inhibition of Human Tumor Cell Growth by BispecificEGFR/c-Met Molecules

Inhibition of human tumor cell growth was assessed in standardattachment culture as described in Examples 3 or 6, or in low attachmentconditions. To assess survival in low attachment conditions, cells wereplated in Ultra Low Attachment 96-well plates (Corning Costar) in 50μL/well of RPMI medium (Invitrogen) containing GlutaMAX and 25 mM Hepes,supplemented with 1 mM sodium pyruvate (Gibco), 0.1 mM NEAA (Gibco), and10% heat inactivated fetal bovine serum (Gibco), and allowed to attachovernight at 37° C., 5% CO₂. Cells were treated with varyingconcentrations of antibodies (0.035-700 nM final), along with HGF (7.5ng/mL, R&D Systems cat #294-HGN), then incubated at 37° C., 5% CO₂ for72 hours. Some wells were left untreated with either HGF or antibodiesas controls. Viable cells were detected using CellTiter-Glo® reagent(Promega), and data were analyzed as described above in “Inhibition ofHuman Tumor Cell Growth (NCI-H292 growth and NCI-H322 growth assay)” inExample 3, except that lysates were transferred to opaque white 96-welltissue culture-treated plates (PerkinElmer) prior to readingluminescence.

Cell line was classified as a strong responder to EGFR/c-Met bispecificmolecule in those instances when maximum inhibition of cell growthwas >40% and relative IC₅₀<5 mM.

Inhibitory activity of ECB15 was assessed in multiple cell lines havingwild type, amplified or mutant EGFR and wild type or amplified c-Met.ECB15 inhibited tumor cell growth of cell lines shown in Table 20. ECB15also inhibited growth of NCI-H1975 cell line having mutation T790M whichhas been show to result in resistance to TKIs such as erlotinib.

TABLE 20 Cell line Histology EGFR c-Met NCI-H1650 Broncho-alveolar- Del(E746, A750) WT adenocarcinoma SKMES-1 Squamous WT WT NCI-H1563Adenocarcinoma GLC-82 Adenocarcinoma Calu-3 Adenocarcinoma NCI-H1573Adenocarcinoma AMP AMP NCI-H1435 NSCLC NCI-H1975 NSCLC L858R; T790M WTNCI-H1666 Broncho-alveolar- adenocarcinoma HCC2935 NSCLC del(E746-T751), S752I HCC4006 Adenocarcinoma del (L747-E749), A750P H292Mucoepidermoid WT WT H322 Adenocarcinoma WT WT HCC827 Adenocarcinoma del(E746, A750); AMP WT H596 Adeno-squamous WT Exon 14 mixed deletion H1869Squamous WT WT WT: wild type AMP: amplified Del: deletion

SEQUENCE LISTING SEQ ID NO: Type Species Description Sequence 1 PRTArtificial Tencon LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT 2 DNA Artificial POP2220GGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGT TTCTGAAGTTACC 3 DNAArtificial TC5′toFG AACACCGTAGATAGAAACGGT 4 DNA Artificial 130merCGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTG 5 DNA Artificial POP2222CGGCGGTTAGAACGCGGCTAC 6 DNA Artificial TCF7GGTGGTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNN AACACCGTAGATAGAAACGGT 7DNA Artificial TCF8 GGTGGTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNSNNAACACCGTAGATAGAAACGGT 8 DNA Artificial TCF9GGTGGTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNSNNSNNAACACCGTAGATAGAAACGGT 9 DNA Artificial TCF10GGTGGTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNAACACCGTAGATAGAAACGGT 10 DNA Artificial TCF11GGTGGTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNAACACCGTAGATAGAAACGGT 11 DNA Artificial TCF12GGTGGTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNAACACCGTAGATAGAAACGGT 12 DNA Artificial POP2234AAGATCAGTTGCGGCCGCTAGACTAGAACCGCTGCCATGGTGATGGTGATGGTGACCGCCGGTGGTGAATTCCGCAGACAG 13 DNA Artificial POP2250CGGCGGTTAGAACGCGGCTACAATTAATAC 14 DNA Artificial DidLigRevCATGATTACGCCAAGCTCAGAA 15 DNA Artificial Tcon5new2GAGCCGCCGCCACCGGTTTAATGGTGATGGTGATGGT GACCACCGGTGGTGAATTCCGCAGACAG 16DNA Artificial Tcon6 AAGAAGGAGAACCGGTATGCTGCCGGCGCCGAAAAAC 17 DNAArtificial LS1008 TTTGGGAAGCTTCTAGGTCTCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCCCAAC TGATCTTCACCAAAC 18 PRT ArtificialP53A1R5- LPAPKNLVVSEVTEDSLRLSWADPHGFYDSFLIQYQES 17 withoutEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV met YKDTNMRGLPLSAEFTT 19 PRTArtificial P54AR4-17 LPAPKNLVVSEVTEDSLRLSVVTYDRDGYDSFLIQYQES without metEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 20 PRTArtificial P54AR4-47 LPAPKNLVVSEVTEDSLRLSWGYNGDHFDSFLIQYQES without metEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 21 PRTArtificial P54AR4-48 LPAPKNLVVSEVTEDSLRLSWDDPRGFYESFLIQYQES without metEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 22 PRTArtificial P54AR4-37 LPAPKNLVVSEVTEDSLRLSVVTWPYADLDSFLIQYQES without metEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 23 PRTArtificial 54AR4-74 LPAPKNLVVSEVTEDSLRLSWGYNGDHFDSFLIQYQES without metEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 24 PRTArtificial P54AR4-81 LPAPKNLVVSEVTEDSLRLSWDYDLGVYFDSFLIQYQE without metSEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHN VYKDTNMRGLPLSAEFTT 25 PRTArtificial P54AR4-83 LPAPKNLVVSEVTEDSLRLSWDDPWAFYESFLIQYQES without metEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 26 PRTArtificial P54CR4-31 LPAPKNLVVSEVTEDSLRLSVVTAPDAAFDSFLIQYQESEwithout Met KVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVLGSY VFEHDVMLPLSAEFTT 27PRT Artificial P54AR4-83v2 LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESwithout Met EKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAIFTT 28PRT Artificial P54CR4-31v2 LPAPKNLVVSEVTEDSARLSVVTAPDAAFDSFLIQYQESEwithout Met KVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVLGSY VFEHDVMLPLSAIFTT 29PRT Artificial P54AR4-73v2 LPAPKNLVVSEVTEDSLRLSVVTWPYADLDSFLIQYQESwihtout Met EKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 30DNA Artificial TCON6 AAG AAG GAG AAC CGG TAT GCT GCC GGC GCC GAA AAA C31 DNA Artificial TCON5 GAG CCG CCG CCA CCG GTT TAA TGG TGA TGG TGAE86Ishort TGG TGA CCA CCG GTG GTG AAG ATC GCA GAC AG 32 PRT ArtificialP114AR5P74- LPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFWIRYDEV A5VVGGEAIVLTVPGSERSYDLTGLKPGTEYYVNILGVKGG SISVPLSAIFTT 33 PRT ArtificialP114AR5P75- LPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFFIRYDEFL E9RSGEAIVLTVPGSERSYDLTGLKPGTEYVVVTILGVKGGL VSTPLSAIFTT 34 PRT ArtificialP114AR7P92- LPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFWIRYFEFL F3GSGEAIVLTVPGSERSYDLTGLKPGTEYIVNIMGVKGGSI SHPLSAIFTT 35 PRT ArtificialP114AR7P92- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL F6GSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGGL SVPLSAIFTT 36 PRT ArtificialP114AR7P92- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIRYFEFLG G8SGEAIVLTVPGSERSYDLTGLKPGTEYVVQILGVKGGYISI PLSAIFTT 37 PRT ArtificialP114AR7P92- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYLEFLL H5GGEAIVLTVPGSERSYDLTGLKPGTEYVVQIMGVKGGTVS PPLSAIFTT 38 PRT ArtificialP114AR7P93- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL D11GSGEAIVLTVPGSERSYDLTGLKPGTEYVVGINGVKGGYI SYPLSAIFTT 39 PRT ArtificialP114AR7P93- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL G8GSGEAIVLTVPGSERSYDLTDLKPGTEYGVTINGVKGGRV STPLSAIFTT 40 PRT ArtificialP114AR7P93- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL H9GSGEAIVLTVPGSERSYDLTGLKPGTEYVVQIIGVKGGHIS LPLSAIFTT 41 PRT ArtificialP114AR7P94- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL A3GSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKI SPPLSAIFTT 42 PRT ArtificialP114AR7P94- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL E5GSGEAIVLTVPGSERSYDLTGLKPGTEYAVNIMGVKGGRV SVPLSAIFTT 43 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL B9GSGEAIVLTVPGSERSYDLTGLKPGTEYVVQILGVKGGSI SVPLSAIFTT 44 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL D3GSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGSI SYPLSAIFTT 45 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL D4GSGEAIVLTVPGSERSYDLTGLKPGTEYVVQILGVKGGYI SIPLSAIFTT 46 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL E3GSGEAIVLTVPGSERSYDLTGLKPGTEYVVQIMGVKGGTV SPPLSAIFTT 47 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFTT F10AGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGSIS PPLSAIFTT 48 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFELLS G7TGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGSIS PPLSAIFTT 49 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFV H8SKGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGSI SPPLSAIFTT 50 PRT ArtificialECB1 MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYDEVVVGGEAIVLTVPGSERSYDLTGLKPGTEYYVNILGVKGGSIS VPLSAIFTT 51 PRT ArtificialECB2 MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKIS PPLSAIFTT 52 PRT ArtificialECB3 MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLIVPGSERSYDLTGLKPGTEYVVQIIGVKGGHIS LPLSAIFTT 53 PRT ArtificialECB4 MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIRYDEFLRSGEAIVLTVPGSERSYDLTGLKPGTEYWVTILGVKGGLVS TPLSAIFTT 54 PRT ArtificialECB5 MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKI SPPLSAIFTT 55 PRT ArtificialECB6 MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLIVPGSERSYDLTGLKPGTEYVVQIIGVKGGHIS LPLSAIFTT 56 PRT ArtificialECB7 MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLIVPGSERSYDLTGLKPGTEYVVQIIGVKGGHIS LPLSAIFTT 57 PRT ArtificialECB15 MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKISPPLSAIFTT 58 PRT Artificial ECB27MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYDEVVVGGEAIVLTVPGSERSYDLTGLKPGTEYYVNILGVKGGSISVPLSAIFTT 59 PRT Artificial ECB60MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKISPPLSAIFTT 60 PRT Artificial ECB37MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYDEVVVGGEAIVLTVPGSERSYDLTGLKPGTEYYVNILGVKGGSISVPLSAIFTT 61 PRT Artificial ECB94MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGKISPPLSAIFTT 62 PRT Artificial ECB95MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 63 PRT Artificial ECB96MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGDAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 64 PRT Artificial ECB97MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT 65 PRT Artificial ECB106MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGKISPPLSAIFTT 66 PRT Artificial ECB107MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 67 PRT Artificial ECB108MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGDAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 68 PRT Artificial ECB109MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT 69 PRT Artificial ECB118MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGKISPPLSAIFTT 70 PRT Artificial ECB119MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 71 PRT Artificial ECB120MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGDAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 72 PRT Artificial ECB121MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTTSEQ ID NO: 73, PRT, Homo Sapiens, EGFR    1mrpsgtagaa llallaalcp asraleekkv cqgtsnkltq lgtfedhfls lqrmfnncev   61vlgnleityv qrnydlsflk tiqevagyvl ialntverip lenlqiirgn myyensyala  121vlsnydankt glkelpmrnl qeilhgavrf snnpalcnve siqwrdivss dflsnmsmdf  181qnhlgscqkc dpscpngscw gageencqkl tkiicaqqcs grcrgkspsd cchnqcaagc  241tgpresdclv crkfrdeatc kdtcpplmly npttyqmdvn pegkysfgat cvkkcprnyv  301vtdhgscvra cgadsyemee dgvrkckkce gperkvcngi gigefkdsls inatnikhfk  361nctsisgdlh ilpvafrgds fthtppldpq eldilktvke itgflliqaw penrtdlhaf  421enleiirgrt kqhgqfslav vslnitslgl rslkeisdgd viisgnknlc yantinwkkl  481fgtsgqktki isnrgensck atgqvchalc spegcwgpep rdcvscrnvs rgrecvdkcn  541llegeprefv enseciqchp eclpqamnit ctgrgpdnci qcahyidgph cvktcpagvm  601genntivwky adaghvchlc hpnctygctg pglegcptng pkipsiatgm vgalllllvv  661algiglfmrr rhivrkrtlr rllqerelve pltpsgeapn qallrilket efkkikvlgs  721gafgtvykgl wipegekvki pvaikelrea tspkankeil deayvmasvd nphvcrllgi  781cltstvqlit qlmpfgclld yvrehkdnig sqyllnwcvq iakgmnyled rrlvhrdlaa  841rnvlvktpqh vkitdfglak llgaeekeyh aeggkvpikw malesilhri ythqsdvwsy  901gvtvwelmtf gskpydgipa seissilekg erlpqppict idvymimvkc wmidadsrpk  961freliiefsk mardpqrylv iqgdermhlp sptdsnfyra lmdeedmddv vdadeylipq 1021qgffsspsts rtpllsslsa tsnnstvaci drnglqscpi kedsflqrys sdptgalted 1081siddtflpvp eyinqsvpkr pagsvqnpvy hnqpinpaps rdphyqdphs tavgnpeyln 1141tvqptcvnst fdspahwaqk gshqisldnp dyqqdffpke akpngifkgs taenaeylry 1201apqssefiga 74 PRT Homo EGF NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGsapiens ERCQYRDLKWWELR SEQ ID NO: 75, PRT, Homo Sapiens, Tenascin-C    1mgamtqllag vflaflalat eggvlkkvir hkrqsgvnat lpeenqpvvf nhvyniklpv   61gsqcsvdles asgekdlapp sepsesfqeh tvdgenqivf thriniprra cgcaaapdvk  121ellsrleele nlvsslreqc tagagcclqp atgrldtrpf csgrgnfste gcgcvcepgw  181kgpncsepec pgnchlrgrc idgqcicddg ftgedcsqla cpsdcndqgk cvngvcicfe  241gyagadcsre icpvpcseeh gtcvdglcvc hdgfagddcn kplclnncyn rgrcvenecv  301cdegftgedc selicpndcf drgrcingtc yceegftged cgkptcphac htqgrceegq  361cvcdegfagv dcsekrcpad chnrgrcvdg rcecddgftg adcgelkcpn gcsghgrcvn  421gqcvcdegyt gedcsqlrcp ndchsrgrcv egkcvceqgf kgydcsdmsc pndchqhgrc  481vngmcvcddg ytgedcrdrq cprdcsnrgl cvdgqcvced gftgpdcael scpndchgqg  541rcvngqcvch egfmgkdcke qrcpsdchgq grcvdgqcic hegftgldcg qhscpsdcnn  601lgqcvsgrci cnegysgedc sevsppkdlv vtevteetvn lawdnemrvt eylvvytpth  661egglemqfry pgdqtstiiq elepgveyfi rvfailenkk sipvsarvat ylpapeglkf  721ksiketsvev ewdpldiafe tweiifrnmn kedegeitks lrrpetsyrq tglapgqeye  781islhivknnt rgpglkrvtt trldapsqie vkdvtdttal itwfkplaei dgieltygik  841dvpgdrttid ltedenqysi gnlkpdteye vslisrrgdm ssnpaketft tgldaprnlr  901rvsqtdnsit lewrngkaai dsyrikyapi sggdhaevdv pksqqattkt tltglrpgte  961ygigvsavke dkesnpatin aateldtpkd lqvsetaets ltllwktpla kfdryrinys 1021lptgqwvgvq lprnttsyvl rglepgqeyn vlltaekgrh kskparvkas teqapelenl 1081tvtevgwdgl rinwtaadqa yehfiiqvqe ankveaarnl tvpgslravd ipglkaatpy 1141tvsiygviqg yrtpvlsaea stgetpnlge vvvaevgwda lklnwtapeg ayeyffiqvq 1201eadtveaaqn ltvpgglrst dlpglkaath ytitirgvtq dfsttplsve vlteevpdmg 1261nitvtevswd alrinwttpd gtydqftiqv qeadqveeah nitvpgslrs meipglragt 1321pytvtlhgev rghstrplav evvtedlpql gdlaysevgw dglrinwtaa dnayehfviq 1381vqevnkveaa qnitlpgslr avdipgleaa tpyrvsiygv irgyrtpvls aeastakepe 1441ignlnvsdit pesfnlswma tdgifetfti eiidsnrlle tveynisgae rtahisglpp 1501stdfivylsg lapsirtkti satattealp llenitisdi npygftvswm asenafdsfl 1561vtvvdsgkll dpqeftlsgt qrklelrgli tgigyevmvs gftqghqtkp lraeivteae 1621pevdnllvsd atpdgfrlsw tadegvfdnf vlkirdtkkq sepleitlla pertrditgl 1681reateyeiel ygiskgrrsq tvsaiattam gspkevifsd itensatvsw raptaqvesf 1741rityvpitgg tpsmvtvdgt ktqtrlvkli pgveylvsii amkgfeesep vsgsfttald 1801gpsglvtani tdsealarwq paiatvdsyv isytgekvpe itrtvsgntv eyaltdlepa 1861teytlrifae kgpqksstit akfttdldsp rdltatevqs etalltwrpp rasvtgyllv 1921yesvdgtvke vivgpdttsy sladlspsth ytakiqalng plrsnmiqti fttigllypf 1981pkdcsqamln gdttsglyti ylngdkaeal evfcdmtsdg ggwivflrrk ngrenfyqnw 2041kayaagfgdr reefwlgldn lnkitaqgqy elrvdlrdhg etafavydkf svgdaktryk 2101lkvegysgta gdsmayhngr sfstfdkdtd saitncalsy kgafwyrnch rvnlmgrygd 2161nnhsqgvnwf hwkghehsiq faemklrpsn frnlegrrkr a 76 PRT Artificial FibconLdaptdlqvtnvtdtsityswtppsatitgyritytpsngpgepkeltvppsstsvtitgltpgveyvvslyalkdnqespplvgtqtt 77 PRT Artificial 10th FN3 domain VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPV of fibronectinQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINY (FN10) RT 78 PRTArtificial Linker GSGS 79 PRT Artificial LinkerGGGGSGGGGSGGGGSGGGGSGGGGS 80 PRT Artificial Linker APAP 81 PRTArtificial Linker APAPAPAPAP 82 PRT Artificial LinkerAPAPAPAPAPAPAPAPAPAP 83 PRT Artificial LinkerAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPA PAP 84 PRT Artificial LinkerAEAAAKEAAAKEAAAKEAAAKEAAAKAAA 85 PRT Artificial Tencon BC loop TAPDAAFD86 PRT Artificial Tencon GF loop KGGHRSN 87 PRT ArtificialP53A1R5-17 BC loop ADPHGFYD 88 PRT Artificial P54AR4-17 BC loop TYDRDGYD89 PRT Artificial P54AR4-47 BC loop WDPFSFYD 90 PRT ArtificialP54AR4-48 BC loop DDPRGFYE 91 PRT Artificial P54AR4-73 BC loop TWPYADLD92 PRT Artificial P54AR4-74 BC loop GYNGDHFD 93 PRT ArtificialP54AR4-81 BC loop DYDLGVYD 94 PRT Artificial P54AR4-83 BC loop DDPWDFYE95 PRT Artificial FG loops of EGFR HNVYKDTNMRGL 96 PRT ArtificialFG loops of EGFR LGSYVFEHDVM 97 DNA Artificial >EGFR part ECB97;Atgttgccagcgccgaagaacctggtagttagcgaggttactgaggac P54AR4-83v22agcgcgcgtctgagctgggacgatccgtgggcgttctacgagagctttctgatccagtatcaagagagcgagaaagtcggtgaagcgattgtgctgaccgtcccgggctccgagcgttcctacgacctgaccggtttgaagccgggtaccgagtatacggtgagcatctacggtgttcacaatgtctataaggacactaatatccgcggtctgcctctgagcgccattttcaccacc 98 DNAArtificial >EGFR part ECB15;Atgctgccagcccctaagaatctggtcgtgagcgaagtaaccgagga P54AR4-83v2cagcgcccgcctgagctgggacgacccgtgggcgttctatgagtctttcctgattcagtatcaagaaagcgaaaaagttggcgaagcgatcgtcctgaccgtcccgggtagcgagcgctcctacgatctgaccggcctgaaaccgggtacggagtacacggtgtccatttacggtgttcacaatgtgtataaagacaccaacatgcgtggcctgccgctgtcggcgattttcaccacc 99 PRT Artificial tencon 27LPAPKNLWSRVTEDSARLSVVTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYG VKGGHRSNPLSAIFTT 100 PRTArtificial TCL14 library LPAPKNLWSRVTEDSARLSVVTAPDAAFDSFXIXYXEXXXXGEAIVLTVPGSERSYDLTGLKPGTEYXVXIXG VKGGXXSXPLSAIFTT >SEQ ID NO: 101PRT Homo sapiens cMet    1mkapavlapg ilvllftivq rsngeckeal aksemnvnmk yqlpnftaet piqnvilheh   61hiflgatnyi yvineedlqk vaeyktgpvl ehpdcfpcqd csskanlsgg vwkdninmal  121vvdtyyddql iscgsvnrgt cqrhvfphnh tadiqsevhc ifspqieeps qcpdcvvsal  181gakvlssvkd rfinffvgnt inssyfpdhp lhsisvrrlk etkdgfmflt dqsyidvlpe  241frdsypikyv hafesnnfiy fltvqretld aqtfhtriir fcsinsglhs ymemplecil  301tekrkkrstk kevfnilqaa yvskpgagla rqigaslndd ilfgvfaqsk pdsaepmdrs  361amcafpikyv ndffnkivnk nnvrclqhfy gpnhehcfnr tllrnssgce arrdeyrtef  421ttalqrvdlf mgqfsevllt sistfikgdl tianlgtseg rfmqvvvsrs gpstphvnfl  481ldshpvspev ivehtlnqng ytivitgkki tkipinglgc rhfqscsqcl sappfvqcgw  541chdkcvrsee clsgtwtqqi clpaiykvfp nsapleggtr lticgwdfgf rrnnkfdlkk  601trvllgnesc tltlsestmn tlkctvgpam nkhfnmsiii snghgttqys tfsyvdpvit  661sispkygpma ggtlltltgn ylnsgnsrhi siggktctlk sysnsilecy tpaqtistef  721avklkidlan retsifsyre dpivyeihpt ksfistwwke pinivsflfc fasggstitg  781vgknlnsysv prmvinvhea grnftvacqh rsnseiicct tpslqqlnlq 1plktkaffm  841ldgilskyfd liyvhnpvfk pfekpvmism gnenvleikg ndidpeavkg evlkvgnksc  901enihlhseav lctvpndllk lnselniewk qaisstvlgk vivqpdqnft gliagvvsis  961talllllgff lwlkkrkqik dlgselvryd arvhtphldr lvsarsyspt temvsnesvd 1021yratfpedqf pnssqngscr qvqypltdms piltsgdsdi sspllqntvh idlsalnpel 1081vqavqhvvig psslivhfne vigrghfgcv yhgtlldndg kkihcavksl nritdigevs 1141qfltegiimk dfshpnvlsl lgiclrsegs plvvlpymkh gdlinfirne thnptvkdli 1201gfglqvakgm kylaskkfvh rdlaarncml dekftvkvad fglardmydk eyysvhnktg 1261aklpvkwmal eslqtqkftt ksdvwsfgvl lwelmtrgap pypdvntfdi tvyllqgrrl 1321lqpeycpdpl yevmlkcwhp kaemrpsfse lvsrisaifs tfigehyvhv natyvnvkcv 1381apypsllsse dnaddevdtr pasfwets 102 PRT Homo HGFQRKRRNTIHEFKKSAKTTLIKIDPALKIK sapiensTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLVVFPFNSMS SGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRG KDLQENYCRNPRGEEGGPVVCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDH TESGKICQRVVDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIK TCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRVVDSQYPHEHDMTPENFKC KDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQT RSGLTCSMWDKNMEDLHRHIFVVEPDASKLNENYCRNPDDDAHGPWCYTGNPLIFVVDYCPIS RCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESW VLTARQCFPSRDLKDYEAVVLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLAR PAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRG KVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIFNRPGIFV RVAYYAKWIHKII LTYKVPQS103 DNA Artificial >cMET part ECB97Ctgccggctccgaagaacttggtggtgagccgtgttaccgaagatagc P114AR7P95-05v2gcacgcctgagctggacggcaccggatgcggcgttcgatagcttctggattcgctattttgagtttctgggtagcggtgaggcaattgttctgacggtgccgggctctgaacgctcctacgatttgaccggtctgaaaccgggcaccgagtatgtggtgaacattctgagcgttaagggcggtagcatcagcccaccgctgagcgcgatcttcacgactggtggttgc 104 DNA Artificial >cMET part ECB15Ctgccggcaccgaagaacctggttgtcagccgtgtgaccgaggatag P114AR7P94-A3cgcacgtttgagctggaccgctccggatgcagcctttgacagcttctggattcgttactttgaatttctgggtagcggtgaggcgatcgttctgacggtgccgggctctgaacgcagctatgatttgacgggcctgaagccgggtactgagtacgtggttaacatcatgggcgttaagggtggtaaaatcagcccgccatt gtccgcgatctttaccacg105 PRT Artificial linker GGGGS 106 PRT Artificial ECB91mlpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltypgsersydltglkpgteytvsiygvhnvykdtnirglplsaifttapapapapapLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT 107 PRT Artificial P53A1R5-17v2lpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnmrglplsaiftt 108 PRT Artificial P54AR4-83v22lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydItglkpgteytvsiygvhnvykdtnirglplsaiftt 109 PRT Artificial P54AR4-83v23lpapknlvvsevtedsarlswddphafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnirglplsaiftt 110 PRT Artificial P53A1R5-17v22lpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnirglplsaiftt 111 PRT ArtificialP114AR7P94-A3v22lpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvnilgvkggkispplsaiftt 112 PRT Artificial P114AR9P121-A6v2LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSGEA1VLIVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 113 PRT ArtificialP114AR9P122-A7v2 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGDAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 114 PRT ArtificialP114AR7P95-05v2 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEA1VLIVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT 115 DNA Artificial ECB97atgttgccagcgccgaagaacctggtagttagcgaggttactgaggacagcgcgcgtctgagctgggacgatccgtgggcgttctacgagagctttctgatccagtatcaagagagcgagaaagtcggtgaagcgattgtgctgaccgtcccgggctccgagcgttcctacgacctgaccggtttgaagccgggtaccgagtatacggtgagcatctacggtgttcacaatgtctataaggacactaatatccgcggtctgcctctgagcgccattttcaccaccgcaccggcaccggctccggctcctgccccgctgccggctccgaagaacttggtggtgagccgtgttaccgaagatagcgcacgcctgagctggacggcaccggatgcggcgttcgatagcttctggattcgctattttgagtttctgggtagcggtgaggcaattgttctgacggtgccgggctctgaacgctcctacgatttgaccggtctgaaaccgggcaccgagtatgtggtgaacattctgagcgttaagggcggtagcatcagcccaccgctgagcgcgatcttcacgactggtggttgc 116 DNA ArtificialECB15 atgctgccagcccctaagaatctggtcgtgagcgaagtaaccgaggacagcgcccgcctgagctgggacgacccgtgggcgttctatgagtctttcctgattcagtatcaagaaagcgaaaaagttggcgaagcgatcgtcctgaccgtcccgggtagcgagcgctcctacgatctgaccggcctgaaaccgggtacggagtacacggtgtccatttacggtgttcacaatgtgtataaagacaccaacatgcgtggcctgccgctgtcggcgattttcaccaccgcgcctgcgccagcgcctgcaccggctccgctgccggcaccgaagaacctggttgtcagccgtgtgaccgaggatagcgcacgtttgagctggaccgctccggatgcagcctttgacagcttctggattcgttactttgaatttctgggtagcggtgaggcgatcgttctgacggtgccgggctctgaacgcagctatgatttgacgggcctgaagccgggtactgagtacgtggttaacatcatgggcgttaagggtggtaaaatcagcccgccattgtccgcgatctttaccacg 117 PRT Artificialalbumin binding tidewllkeakekaieelkkagitsdyyfdlinkaktvegvnalkdeilkadomain 118 PRT Artificial ECB18nnlpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwirydevvvggeaivltvpgsersydltglkpgteyyvnilgvkggsisvplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 119 PRT Artificial ECB28nnlpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwirydevvvggeaivltvpgsersydltglkpgteyyvnilgvkggsisvplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 120 PRT Artificial ECB38nnlpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivItvpgsersydItglkpgteyvvnimgvkggkispplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 121 PRT Artificial ECB39nnlpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivItvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivItvpgsersydltglkpgteyvvnimgvkggkispplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 122 PRT ArtificialP53A1R5-17 wth Met MLPAPKNLVVSEVTEDSLRLSWADPHGFYDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 123 PRTArtificial P54AR4-17 with Met MLPAPKNLWSEVTEDSLRLSWTYDRDGYDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 124 PRTArtificial P54AR4-47 with Met MLPAPKNLWSEVTEDSLRLSWGYNGDHFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 125 PRTArtificial P54AR4-48 with Met MLPAPKNLVVSEVTEDSLRLSWDDPRGFYESFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 126 PRTArtificial P54AR4-73 with Met MLPAPKNLWSEVTEDSLRLSWTVVPYADLDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 127 PRTArtificial 54AR4-74 with Met MLPAPKNLWSEVTEDSLRLSWGYNGDHFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 128 PRTArtificial P54AR4-81 with Met MLPAPKNLVVSEVTEDSLRLSWDYDLGVYFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSI YGVHNVYKDTNMRGLPLSAEFTT 129 PRTArtificial P54AR4-83 with Met MLPAPKNLVVSEVTEDSLRLSWDDPWAFYESFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 130 PRTArtificial P540R4-31 with Met MLPAPKNLWSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVLGSYVFEHDVMLPLSAEFTT 131 PRTArtificial P54AR4-83v2 with MLPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQY MetQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAIFTT 132 PRTArtificial P540R4-31v2 with MLPAPKNLVVSEVTEDSARLSWTAPDAAFDSFLIQY MetQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVLGSYVFEHDVMLPLSAIFTT 133 PRTArtificial P54AR4-73v2 MLPAPKNLWSEVTEDSLRLSWTWPYADLDSFLIQY withMetQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 134 PRTArtificial P53A1R5-17v2 withmlpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgser Metsydltglkpgteytvsiygvhnvykdtnmrglplsaiftt 135 PRT ArtificialP54AR4-83v22 with mlpapknIwsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgseMet rsydltglkpgteytvsiygvhnvykdtnirglplsaiftt 136 PRT ArtificialP54AR4-83v23 withmlpapknlvvsevtedsarlswddphafyesfliqyqesekvgeaivltvpgser Metsydltglkpgteytvsiygvhnvykdtnirglplsaiftt 137 PRT ArtificialP53A1R5-17v22 withmlpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgser Metsydltglkpgteytvsiygvhnvykdtnirglplsaiftt 138 PRT ArtificialECB1 without Met LPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSMLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYDEVVVGGEAIVLTVPGSERSYDLTGLKPG TEYYVNILGVKGGSISVPLSAIFTT 139 PRTArtificial ECB2 without Met LPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGT EYWNIMGVKGGKISPPLSAIFTT 140 PRTArtificial ECB3 without Met LPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSMLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPG TEYWQIIGVKGGHISLPLSAIFTT 141 PRTArtificial ECB4 without Met LPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSMLPAPKNLWSRVTEDSARLSWTAPDAAFDSFFIRYDEFLRSGEAIVLTVPGSERSYDLTGLKPGT EYWVTILGVKGGLVSTPLSAIFTT 142 PRTArtificial ECB5 without Met LPAPKNLWSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSMLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPG TEYWNIMGVKGGKISPPLSAIFTT 143 PRTArtificial ECB6 without Met LPAPKNLWSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSMLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPG TEYWQIIGVKGGHISLPLSAIFTT 144 PRTArtificial ECB7 without Met LPAPKNLWSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSMLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPG TEYWQIIGVKGGHISLPLSAIFTT 145 PRTArtificial ECB15 without Met LPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSVVTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYWNIMGVKGGKI SPPLSAIFTT 146 PRT ArtificialECB27 without Met LPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSVVTAPDAAFDSFWIRYDEVVVGGEAIVLTVPGSERSYDLTGLKPGTEYYVNILGVKGGSI SVPLSAIFTT 147 PRT ArtificialECB60 without Met LPAPKNLWSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPMLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGS GEAIVLTVPGSERSYDLTGLKPGTEYWNIMGVKGGKISPPLSAIFTT 148 PRT Artificial ECB37 without MetLPAPKNLWSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSVVTAPDAAFDSFWIRYDEVVVGGEAIVLTVPGSERSYDLTGLKPGTEYYVNILGVKGGSI SVPLSAIFTT 149 PRT ArtificialECB94 without Met LPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGE AIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGKISPPLSAIFTT 150 PRT Artificial ECB95 without MetLPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSG EAIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGSISPPLSAIFTT 151 PRT Artificial ECB96 without MetLPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGD AIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGSISPPLSAIFTT 152 PRT Artificial ECB97 without MetLPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYWNILSVKGGSISP PLSAIFTT 153 PRT ArtificialECB106 without Met LPAPKNLWSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGE AIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGKISPPLSAIFTT 154 PRT Artificial ECB107 without MetLPAPKNLWSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSG EAIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGSISPPLSAIFTT 155 PRT Artificial ECB108 without MetLPAPKNLWSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGD AIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGSISPPLSAIFTT 156 PRT Artificial ECB109 without MetLPAPKNLWSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYWNILSVKGGSISP PLSAIFTT 157 PRT ArtificialECB118 without Met LPAPKNLWSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGE AIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGKISPPLSAIFTT 158 PRT Artificial ECB119 without MetLPAPKNLWSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSG EAIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGSISPPLSAIFTT 159 PRT Artificial ECB120 without MetLPAPKNLWSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGD AIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGSISPPLSAIFTT 160 PRT Artificial ECB121 without MetLPAPKNLWSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYWNILSVKGGSISP PLSAIFTT 161 PRT ArtificialECB91 without MetlpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltypgsersydltglkpgteytysiygvhnvykdtnirglplsaifttapapapapapLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT 162 PRT ArtificialECB18 without Met lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwirydevvvggeaivItvpgsersydltglkpgteyyvnilgvkggsisvplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 163 PRT ArtificialECB28 without Met lpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwirydevvvggeaivltvpgsersydltglkpgteyyvnilgvkggsisvplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 164 PRT ArtificialECB38 without Met lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvnimgvkggkispplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 165 PRT ArtificialECB39 without Met lpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvnimgvkggkispplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 166 DNA ArtificialECB97 without Met ttgccagcgccgaagaacctggtagttagcgaggttactgaggacagcgcgcgtctgagctgggacgatccgtgggcgttctacgagagctttctgatccagtatcaagagagcgagaaagtcggtgaagcgattgtgctgaccgtcccgggctccgagcgttcctacgacctgaccggtttgaagccgggtaccgagtatacggtgagcatctacggtgttcacaatgtctataaggacactaatatccgcggtctgcctctgagcgccattttcaccaccgcaccggcaccggctccggctcctgccccgctgccggctccgaagaacttggtggtgagccgtgttaccgaagatagcgcacgcctgagctggacggcaccggatgcggcgttcgatagcttctggattcgctattttgagtttctgggtagcggtgaggcaattgttctgacggtgccgggctctgaacgctcctacgatttgaccggtctgaaaccgggcaccgagtatgtggtgaacattctgagcgttaagggcggtagcatcagcccaccgctgagcgcgatcttcacgactggtggttgc 167 DNA ArtificialECB15 without Met ctgccagcccctaagaatctggtcgtgagcgaagtaaccgaggacagcgcccgcctgagctgggacgacccgtgggcgttctatgagtctttcctgattcagtatcaagaaagcgaaaaagttggcgaagcgatcgtcctgaccgtcccgggtagcgagcgctcctacgatctgaccggcctgaaaccgggtacggagtacacggtgtccatttacggtgttcacaatgtgtataaagacaccaacatgcgtggcctgccgctgtcggcgattttcaccaccgcgcctgcgccagcgcctgcaccggctccgctgccggcaccgaagaacctggttgtcagccgtgtgaccgaggatagcgcacgtttgagctggaccgctccggatgcagcctttgacagcttctggattcgttactttgaatttctgggtagcggtgaggcgatcgttctgacggtgccgggctctgaacgcagctatgatttgacgggcctgaagccgggtactgagtacgtggttaacatcatgggcgttaagggtggtaaaatcagcccgccattgtccgcgatctttaccacg 168 DNAArtificial >EGFR part ECB97;ttgccagcgccgaagaacctggtagttagcgaggttactgaggacagc P54AR4-83v22gcgcgtctgagctgggacgatccgtgggcgttctacgagagctttctgat without metccagtatcaagagagcgagaaagtcggtgaagcgattgtgctgaccgtcccgggctccgagcgttcctacgacctgaccggtttgaagccgggtaccgagtatacggtgagcatctacggtgttcacaatgtctataaggacactaatatccgcggtctgcctctgagcgccattttcaccacc 169 DNAArtificial >EGFR part ECB15;ctgccagcccctaagaatctggtcgtgagcgaagtaaccgaggacag P54AR4-83v2cgcccgcctgagctgggacgacccgtgggcgttctatgagtctttcctga without Metttcagtatcaagaaagcgaaaaagttggcgaagcgatcgtcctgaccgtcccgggtagcgagcgctcctacgatctgaccggcctgaaaccgggtacggagtacacggtgtccatttacggtgttcacaatgtgtataaagacaccaacatgcgtggcctgccgctgtcggcgattttcaccacc 170 PRT ArtificialECB94 with C-ter MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGKI SPPLSAIFTTC 171 PRT ArtificialECB95 with C-ter MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSI SPPLSAIFTTC 172 PRT ArtificialECB96 with C-ter MLPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKG DAIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGSISPPLSAIFTTC 173 PRT Artificial ECB97 with C-terMLPAPKNLWSEVTEDSARLSWDDPWAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYWNILSVKGGSIS PPLSAIFTTC 174 PRT ArtificialECB106 with C-ter MLPAPKNLWSEVTEDSARLSWDDPHAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSG EAIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGKISPPLSAIFTTC 175 PRT Artificial ECB107 with C-terMLPAPKNLWSEVTEDSARLSWDDPHAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSG EAIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGSISPPLSAIFTTC 176 PRT Artificial ECB108 with C-terMLPAPKNLWSEVTEDSARLSWDDPHAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKG DAIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGSISPPLSAIFTTC 177 PRT Artificial ECB109 with C-terMLPAPKNLWSEVTEDSARLSWDDPHAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYWNILSVKGGSIS PPLSAIFTTC 178 PRT ArtificialECB91 with C-ter mlpapknlwsevtedsarlswddpwafyesfliqyqesekvgeaivltypgsecysteine rsydltglkpgteytysiygvhnvykdtnirglplsaifttapapapapapLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTTC >SEQ ID NO: 179 PRT ArtificialAn FG loop of EGFR binding FN3 domain HNVYKDTNX₉RGL;wherein X₉ is M or I >SEQ ID NO: 180 PRT ArtificialA FG loop of EGFR binding FN3 domainLGSYVFEHDVML (SEQ ID NO: 180), >SEQ ID NO: 181 PRT Artificiala BC loop of EGFR binding FN3 domainX₁X₂X₃X₄X₅X₆X₇X₈(SEQ ID NO: 181), wherein X₁ is A, T, G or D;X₂ is A, D, Y or W; X₃ is P, D or N; X₄ is L or absent;X₅ is D, H, R, G, Y or W; X₆ is G, D or A; X₇ is A, F, G, H or D; andX₈ is Y, F or L. >SEQ ID NO: 182 PRT Artificial EGFR binding FN3 domainLPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNX₉RGLPLSAEFTT (SEQ ID NO: 182),X₁ is A, T, G or D; X₂ is A, D, Y or W; X₃ is P, D or N;X₄ is L or absent; X₅ is D, H, R, G, Y or W; X₆ is G, D or A;X₇ is A, F, G, H or D; X₈ is Y, F or L; and X₉ is M or I >SEQ ID NO: 183PRT Artificial EGFR binding FN3 domainLPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVLGSYVFEHDVMLPLSAEFTT (SEQ ID NO: 183), whereinX₁ is A, T, G or D; X₂ is A, D, Y or W; X₃ is P, D or N;X₄ is L or absent; X₅ is D, H, R, G, Y or W; X₆ is G, D or A;X₇ is A, F, G, H or D; and X₈ is Y, F or L. >SEQ ID NO: 184 PRTArtificial A C-met binding FN3 domain C strand and a CD loop sequenceDSFX₁₀IRYX₁₁EX₁₂X₁₃X₁₄X₁₅GX₁₆ (SEQ ID NO: 184), whereinX₁₀ is W, F or V; X₁₁ is D, F or L; X₁₂ is V, F or L; X₁₃ is V, L or T;X₁₄ iS V, R, G, L, T or S; X₁₅ is G, S, A, T or K; andX₁₆ is E or D; and >SEQ ID NO: 185 PRT ArtificialA c-Met binding FN3 domain F strand and a FG loopTEYX₁₇VX₁₈IX₁₉X₂₀VKGGX₂₁X₂₂SX₂₃ (SEQ ID NO: 185), whereinX₁₇ is Y, W, I, V, G or A; X₁₈ is N, T, Q or G; X₁₉ is L, M, N or I;X₂₀ is G or S; X₂₁ is S, L, G, Y, T, R, H or K; X₂₂ is I, V or L; andX₂₃ is V, T, H, I, P, Y or L. >SEQ ID NO: 186 PRT Artificiala c-Met binding FN3 domainLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX₁₀IRYX₁₁EX₁₂X₁₃X₁₄X₁₅GX₁₆AIVLTVPGSERSYDLTGLKPGTEYX₁₇VX₁₈IX₁₉X₂₀VKGGX₂₁X₂₂SX₂₃PLSAEFTT(SEQ ID NO: 186), wherein X₁₀ is W, F or V; and X₁₁ is D, F or L;X₁₂ is V, F or L; X₁₃ is V, L or T; X₁₄ iS V, R, G, L, T or S;X₁₅ is G, S, A, T or K; X₁₆ is E or D; X₁₇ is Y, W, I, V, G or A;X₁₈ is N, T, Q or G; X₁₉ is L, M, N or I; X₂₀ is G or S;X₂₁ is S, L, G, Y, T, R, H or K; X₂₂ is I, V or L; andX₂₃ is V, T, H, I, P, Y or L. >SEQ ID NO: 187 PRT ArtificialEGFR FN3 domain of a bispecific EGFR/c-Met FN3 domain containing moleculeLPAPKNLVVSX₂₄VTX₂₅DSX₂₆RLSWDDPX₂₇AFYX₂₈SFLIQYQX₂₉SEKVGEAIX₃₀LTVPGSERSYDLTGLKPGTEYTVSIYX₃₁VHNVYKDTNX₃₂RGLPLSAX₃₃FTT (SEQ IDNO: 187), wherein X₂₄ is E, N or R; X₂₅ is E or P; X₂₆ is L or A;X₂₇ is H or W; X₂₈ is E or D; X₂₉ is E or P; X₃₀ is N or V;X₃₁ is G or Y; X₃₂ is M or I; and X₃₃ is E or I; >SEQ ID NO: 188c-Met FN3 domain of a bispecific EGFR/c-Met FN3 domain containing moleculeLPAPKNLVVSX₃₄VTX₃₅DSX₃₆RLSWTAPDAAFDSFWIRYFX₃₇FX₃₈X₃₉X₄₀GX₄₁AIX₄₂LTVPGSERSYDLTGLKPGTEYVVNIX₄₃X₄₄VKGGX₄₅ISPPLSAX₄₆FTT (SEQ ID NO:188); wherein X₃₄ is E, N or R; X₃₅ is E or P; X₃₆ is L or A;X₃₇ is E or P; X₃₈ is V or L; X₃₉ is G or S; X₄₀ is S or K;X₄₁ is E or D; X₄₂ is N or V; X₄₃ is L or M; X₄₄ is G or S;X₄₅ is S or K; and X₄₆ is E or I. >SEQ ID NO: 189 HSA variant C34SDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELGEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL >SEQ ID NO: 190 ECB168MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGFAIVLTVPGSERSYDLTGLKPGIEYVVNILSVKGGSISPPLSAIFTT >SEQ ID NO: 191 ECB168 without Met  LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT >192ECB no name last in the list 17v2-C5v2 mLpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvh nvykdtnmrglplsaifttapapapapap LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPG SERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT  >193ECB no name without met last in the list 17v2-C5v2 Lpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdtnmrglplsaifttapapapapap LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPG SERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT  >SEQ ID NO: 194  83v2 D22A Lpapknlvvsevtedsarlswadpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdtnmrglplsaiftt  >SEQ ID NO: 195  >83v2 D23A Lpapknlvvsevtedsarlswdapwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdtnmrglplsaiftt  >SEQ ID NO: 196  >83v2 P24A Lpapknlvvsevtedsarlswddawafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdtnmrglplsaiftt  >SEQ ID NO: 197  >83v2 W25A Lpapknlvvsevtedsarlswddpaafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdtnmrglplsaiftt  >SEQ ID NO: 198  >83v2 F27A Lpapknlvvsevtedsarlswddpwaayesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdtnmrglplsaiftt  >SEQ ID NO: 199  >83v2 Y28A Lpapknlvvsevtedsarlswddpwafaesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdtnmrglplsaiftt  >SEQ ID NO:200  >83v2 H75A Lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvan vykdtnmrglplsaiftt  >SEQ ID NO: 201  >83v2 N76A Lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvha vykdtnmrglplsaiftt  >SEQ ID NO: 202  >83v2 V77a Lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn aykdtnmrglplsaiftt  >SEQ ID NO: 203  >83v2 Y78A Lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vakdtnmrglplsaiftt  >SEQ ID NO: 204  >83v2 K79A Lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vyadtnmrglplsaiftt  >SEQ ID NO: 205  >83v2 D80A Lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykatnmrglplsaiftt  >SEQ ID NO: 206  >83v2 M83A Lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdtnarglplsaiftt  >SEQ ID NO: 207  >83v2 R84A Lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdtnmaglplsaiftt  >SEQ ID NO: 208  >83v2 G85A Lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdtnmralplsaiftt  >SEQ ID NO: 209  >83v2 L86A Lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdtnmrgaplsaiftt  >SEQ ID NO: 210  >83v2 T81A Lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdanmrglplsaiftt  >SEQ ID NO: 211  >83v2 N82A Lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhn vykdtamrglplsaiftt  SEQ ID NO: 212  >K78S Lpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvnimgvkgg Sispplsaiftt  SEQ ID NO: 213  >G40S LpapknlvvsrvtedsarlswtapdaafdsfwiryfeflSsgeaivltvpgsersydltglkpgteyvvnimgvkgg kispplsaiftt  SEQ ID NO: 214  >L39S LpapknlvvsrvtedsarlswtapdaafdsfwiryfefSgsgeaivltvpgsersydltglkpgteyvvnimgvkg gkispplsaiftt  SEQ ID NO: 215  >V68SLpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteySvnimgvkgg kispplsaiftt  SEQ ID NO: 216  >N70SLpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvSimgvkgg kispplsaiftt  SEQ ID NO: 217  >P81SLpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvnimgvkgg kisSplsaiftt  SEQ ID NO: 218  >F36S lpapknlvvsrvtedsarlswtapdaafdsfwirySeflgsgeaivltvpgsersydltglkpgteyvvnimgvkgg kispplsaiftt  SEQ ID NO: 219  >W32SlpapknlvvsrvtedsarlswtapdaafdsfSiryfeflgsgeaivltvpgsersydltglkpgteyvvnimgvkgg kispplsaiftt  SEQ ID NO: 220  >M725 lpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvniSgvkgg kispplsaiftt  SEQ ID NO: 221  >R34S lpapknlvvsrvtedsarlswtapdaafdsfwiSyfeflgsgeaivltvpgsersydltglkpgteyvvnimgvkgg kispplsaiftt  SEQ ID NO: 222  >F38S lpapknlvvsrvtedsarlswtapdaafdsfwiryfeSlgsgeaivltvpgsersydltglkpgteyvvnimgvkgg kispplsaiftt  SEQ ID NO: 223  >I79S lpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvnimgvkgg kSspplsaiftt  SEQ ID NO: 224  PRT  Artificial  Linker  GGGGSGGGGS 

What is claimed:
 1. A protein comprising a peptide having the amino acidsequence of SEQ ID NO:
 61. 2. The protein of claim 1, further comprisinga cysteine linked to the C-terminus of the protein.
 3. The protein ofclaim 1, further comprising a half-life extending moiety coupled to theprotein.
 4. The protein of claim 3, wherein the half-life extendingmoiety is an albumin binding molecule, a polyethylene glycol (PEG),albumin, or at least a portion of an Fc region of an immunoglobulin. 5.A pharmaceutical composition comprising the protein of claim 1 and apharmaceutically acceptable carrier.
 6. A pharmaceutical compositioncomprising the protein of claim 4 and a pharmaceutically acceptablecarrier.